Stabilized steviol glycoside compositions and uses thereof

ABSTRACT

It has been surprisingly found that the presence of steviol glycoside stabilizing compounds, such as the solubility enhancers described herein, significantly increases the stability of steviol glycosides under most conditions, including highly acidic conditions (e.g., at a pH less than 2, such as the conditions to which SGs might be exposed to, in use, in a throw syrup) and/or at elevated a temperatures (e.g., at temperatures exceeding 25° C., such as at 40° C.).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/569,279, filed Oct. 6, 2017, and entitled “Steviol GlycosideSolubility Enhancers,” and U.S. Provisional Application Ser. No.62/676,722, filed May 25, 2018, and entitled “Methods for Making YerbaMate Extract Composition,” both of which applications are herebyincorporated by reference as if fully set forth herein in theirentirety.

BACKGROUND

Steviol glycosides (SGs) are currently being investigated as sweeteningagents for use in foods, beverages, pharmaceuticals, and oralhygiene/cosmetic products, including in beverages such as carbonatedsoft drinks. The recent discovery of chlorogenic acids and cynarinisomers as solubility enhancers (SEs) for SGs is opening product linesthat were previously unattainable, such as concentrated throw syrups andfountain drinks. While steviol glycosides can provide a non-caloricoption for sweetening such products, there can be challenges topreparing such products with steviol glycosides. Though they can bestable at neutral pH, in some cases, steviol glycoside compositions canhave limited chemical stability, especially at higher concentrations,over longer storage times at low pH and/or elevated temperatures. Forexample, beverage concentrates such as fountain syrups or throw syrupsinherently have a low pH and can require storage over time, sometimes atelevated temperatures.

SUMMARY

The disclosure provides, among other things, the use of steviolglycoside stabilizing compounds to enhance the chemical stability ofSGs, such that the SGs can be used to prepare compositions withincreased chemical stability, especially at higher SG concentrations andover longer storage times. The steviol glycoside stabilizing compoundscan also enhance the chemical stability of SGs under acidic conditionsand/or at elevated temperatures. This disclosure also describes theenhanced chemical stability of the steviol glycoside stabilizingcompounds themselves, which are less subject to acidic hydrolysis and/oroxidation in the presence of SGs. This effect appears to beconcentration dependent, with higher concentrations yielding greaterstability. This finding opens up the possibility of using SGs in waterenhancers, coffee syrups, and liquid stevia products where the SGconcentration is higher, the desired shelf-life is longer, and/or the pHcan be acidic.

While not wishing to be bound by any theory, it is hypothesized that themechanism by which the steviol glycoside stabilizing compounds increasethe chemical stability of SGs in solution has to do, in part, byreducing or altering the interactions between water molecules and SGmolecules. It is also hypothesized that the interactions with strongacids are also reduced, effectively shielding the SGs from acidichydrolysis.

DESCRIPTION OF THE DRAWINGS

The drawings illustrate generally, by way of example, but not by way ofimitation, various embodiments discussed herein.

FIG. 1 is a flow diagram of an example of a method for making acomposition comprising steviol glycoside stabilizing compounds, such ascaffeic acid, monocaffeoylquinic acids, and dicaffeoylquinic acids, andsalts thereof.

FIG. 2 is a flow diagram of another example of a method for making acomposition comprising steviol glycoside stabilizing compounds, such ascaffeic acid, monocaffeoylquinic acids, and dicaffeoylquinic acids, andsalts thereof.

FIG. 3 is a flow diagram of another example of a method for making acomposition comprising steviol glycoside stabilizing compounds, such ascaffeic acid, monocaffeoylquinic acids, and dicaffeoylquinic acids, andsalts thereof.

FIG. 4 is a flow diagram of another example of a method for making acomposition comprising steviol glycoside stabilizing compounds, such ascaffeic acid, monocaffeoylquinic acids, and dicaffeoylquinic acids, andsalts thereof.

Repeated use of reference characters in the specification and drawingsis intended to represent the same or analogous features or elements ofthe disclosure, even when the numbers increase by 100 fromfigure-to-figure (e.g., drying operation 120 in FIG. 1 is analogous toor the same as drying operations 220, 320, and 420 in FIGS. 2-4,respectively). It should be understood that numerous other modificationsand examples can be devised by those skilled in the art, which fallwithin the scope and spirit of the principles of the disclosure.

DESCRIPTION

Reference will now be made in detail to certain embodiments of thedisclosed subject matter, examples of which are illustrated in part inthe accompanying drawings. While the disclosed subject matter will bedescribed in conjunction with the enumerated claims, it will beunderstood that the exemplified subject matter is not intended to limitthe claims to the disclosed subject matter.

The disclosure relates generally to compositions comprising a steviolglycoside (SG) and a steviol glycoside stabilizing compound in an amounteffective to reduce degradation of the SG. The compositions comprisingthe SG and the steviol glycoside stabilizing compound can be formulatedin any suitable way, including as an aqueous composition.

It has been surprisingly found that the presence of steviol glycosidestabilizing compounds significantly increases the stability of SGs undermost conditions, including at higher concentrations of SG and overlonger storage times. Additionally, the presence of steviol glycosidestabilizing compounds significantly increases the stability of SGs underacidic conditions and/or at elevated temperatures. For example, in thepresence of the steviol glycoside stabilizing compounds describedherein, one can not only access SG concentrations of up to 35 wt. %(e.g., from about 1 wt. % to about 35 wt. %, with 5 wt. % being asuitable concentration for a liquid stevia application), but the SGswill be chemically stable at acidic pH over a period of greater than 72days, or even for a period of one year or longer. In sum, the SG/steviolglycoside stabilizing compound compositions are chemically stable forweeks, months or even years, even at acidic pHs and/or elevatedtemperatures.

The term “chemical stability” refers to a reduced chemical degradationof SGs, including hydrolysis and isomerization of the double bond on thesteviol core. For example, a chemically stable Rebaudioside M (Reb M)resists degradation to hydrolysis products such as Rebaudioside A (RebA), Rebaudioside B (Reb B), iso-Rebaudioside M (iso-Reb M),iso-Rebaudioside A (iso-Reb A), and iso-Rebaudioside B (iso-Reb B).Chemical stability can be measured by known methods such as by UHPLCanalysis. For example, as described in the Examples herein, acomposition comprising Reb M can be injected directly for analysis byUHPLC-UV. The chromatographic analysis can be performed on a C18-basedreversed-phase chromatography column at elevated temperature undergradient conditions, utilizing trifluoroacetic acid in water andacetonitrile. SGs can be detected utilizing a UV detector set to 210 nm.A linear calibration curve can be applied using Reb A standard as areference solution.

In some aspects, the amount of steviol glycoside stabilizing compoundeffective to reduce degradation of SG is an amount to chemicallystabilize the SG over higher concentrations of SG and/or over longerstorage times. For example, the amount of SG and/or steviol glycosidestabilizing compound can be at least 1 wt %, at least 3 wt. %, at least5 wt %, at least 10 wt %, at least 15 wt %, at least 20 wt %, at least30 wt. %; about 1 wt % to about 35 wt. %, about 5 wt. % to about 15 wt%, about 1 wt. % to about 5 wt % or about 5 wt. % to about 20 wt %,about And within these stated ranges, the SG and the steviol glycosidestabilizing compound can be chemically stable for days, weeks, months oryears.

In some aspects, the amount of steviol glycoside stabilizing compoundeffective to reduce degradation of SG can be determined by anaccelerated chemical stability assay. For example, the “amount ofsteviol glycoside stabilizing compound effective to reduce degradationof the steviol glycoside” is an amount such that at least about 10%(e.g., at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%; or from about 10% to about100%, about 20% to about 80%, about 30% to about 95%, about 40% to about80%, about 60% to about 90% or about 70% to about 99% or more) relativeto an initial steviol glycoside concentration remains when thestabilized steviol glycoside composition is subjected to storage for 7days at 40° C. in a 5% phosphoric acid solution. In some cases, there isstatistically less degradation of the steviol glycosides than whensteviol glycoside stabilizing compound is absent.

The compositions comprising a SG and a steviol glycoside stabilizingcompound can have a pH of less than about 5 (e.g., less than about 4,less than about 3, less than about 2.5, less than about 2, less thanabout 1.7, less than about 1.5, less than about 1, much less than about1; about 0.1 to about 4, about 1 to about 4, about 0.5 to about 2 orabout 1 to about 3).

The compositions comprising a SG and a steviol glycoside stabilizingcompound are storable at room temperature, at below room temperature(e.g., 4° C.) or at above room temperature (e.g., at 40° C.), withoutany substantial degradation of the steviol glycoside.

The compositions comprising a SG and a steviol glycoside stabilizingcompound can comprise any suitable steviol glycoside, such asstevioside, Reb A, Reb C, dulcoside A, Reb M, Reb B, Reb D, and Reb Eand salts thereof (in cases where compounds can form salts, such as inthe case of Reb B, steviolbioside, and steviol-13-O-glucoside (13-SMG)).The steviol glycoside can be Reb M, Reb A or mixtures of one or more ofthe aforementioned steviol glycosides. In one aspect, the steviolglycoside comprises one or more steviol glycosides selected from thelist consisting of Reb A, Reb B, Reb C, Reb D, Reb E, Reb F, Reb M, RebN, Reb O, rubusoside, dulcoside A, Reb I, Reb Q, 1,2-stevioside,1,3-stevioside, steviol-1,2-bioside, steviol-1,3-bioside, 13-SMG,steviol-19-O-glucoside (19-SMG), and steviol glycosides with 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more sugar additions, such as glucose, rhamnose, orxylose as three examples.

The compositions comprising a SG and a steviol glycoside stabilizingcompound can comprise any suitable steviol glycoside at any suitableconcentration. For example, the compositions comprising a SG and asteviol glycoside stabilizing compound can comprise any suitable amountof steviol glycoside, such as at least about 0.03 wt. % (e.g., at leastabout 0.1 wt. %, at least about 0.5 wt. %, at least about 0.6 wt. %, atleast about 1 wt. %, at least about 1.5 wt. %, at least about 2.5 wt. %,at least 3 wt. %, at least about 5 wt. %, at least about 6 wt. %, atleast about 10 wt. %, at least about 15 wt. %, at least about 20 wt. %,at least about 25 wt. %, at least about 30 wt. %, at least about 35 wt.%, or up to about 40 wt. %; about 0.03 wt. % to about 5 wt. %, about 0.1wt. % to about 10 wt. %, about 1 wt. % to about 4 wt. %, about 0.1 wt. %to about 5 wt. %, about or about 1 wt. % to about 5 wt. %) steviolglycoside.

The compositions comprising a SG and a steviol glycoside stabilizingcompound can comprise any suitable steviol glycoside at any suitableconcentration. The compositions comprising a SG and a steviol glycosidestabilizing compound can comprise concentrated products that are dilutedby the consumer for consumer use. For example, beverage concentratessuch as throw syrups are diluted six to seven times for use and flavoredwater enhancer liquids are diluted up to 100 times.

For example, for a beverage concentrate product comprising thecompositions comprising a SG and a steviol glycoside stabilizingcompound can comprise between about 1,800 ppm and about 10,000 ppm(e.g., about 2,000 ppm to about 5,000 ppm, about 2,000 ppm to about8,000 ppm, about 3,000 ppm to about 5,000 ppm or about 2,500 ppm toabout 7,500 ppm) steviol glycoside.

In another example, this time for a liquid water enhancer product, thecompositions comprising a SG and a steviol glycoside stabilizingcompound can comprise between about 1.5 wt. % and about 3.5 wt. %.(e.g., about 2 wt. % to about 3 wt. %, about 1.5 wt. % to about 2 wt. %or about 2.5 wt. % to about 3.5 wt. %) steviol glycoside.

In yet another example, this time for a liquid sweetener, thecompositions comprising a SG and a steviol glycoside stabilizingcompound can comprise between about 1.0 wt. % and about 10 wt. % (e.g.,about 4 wt. % to about 10 wt. %, about 5 wt. % to about 10 wt. %, about7 wt. % to about 9 wt. % or about 8 wt. % to about 10 wt. %) steviolglycoside.

In some aspects, the compositions comprising a SG and a steviolglycoside stabilizing compound can comprise any suitable additivesincluding buffering agent, acidulants, such as citric acid,antimicrobial agents, such as benzoic acid and sorbic acid (and saltsthereof), natural colors, natural flavors, artificial flavors,artificial colors, and artificial sweeteners.

Examples of steviol glycoside stabilizing compounds include:

caffeic acid, an ester of caffeic acid, an ester of caffeic acid andquinic acid, an ester of caffeic acid and quinic acid comprising asingle caffeic acid moiety (e.g., chlorogenic, cryptochlorogenic, andneochlorogenic acid; structures of each are provided herein), an esterof caffeic acid and quinic acid comprising more than one caffeic acidmoiety (e.g., 1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid,1,5-dicaffeoylqunic acid, 3,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid; structures ofeach are provided herein);

ferulic acid, an ester of ferulic acid, an ester of ferulic acid andquinic acid, an ester of ferulic acid and quinic acid comprising asingle ferulic acid moiety, an ester of ferulic acid and quinic acidcomprising more than one ferulic acid moiety;

3-(3,4-dihydroxyphenyl) lactic acid, a 3-(3,4-dihydroxyphenyl)lacticacid derivative, an ester of 3-(3,4-dihydroxyphenyl)lactic acid, anester of a 3-(3,4-dihydroxyphenyl)lactic acid derivative,

quinic acid, a quinic acid derivative, an ester of quinic acid, an esterof a quinic acid derivative;

p-coumaric acid, an ester of p-coumaric acid, an ester of p-coumaricacid and quinic acid, an ester of p-coumaric acid and quinic acidcomprising a single p-coumaric acid moiety, an ester of p-coumaric acidand quinic acid comprising more than one p-coumaric acid moiety;

sinapic acid, an ester of sinapic acid, an ester of sinapic acid andquinic acid, an ester of sinapic acid and quinic acid comprising asingle sinapic acid moiety, an ester of sinapic acid and quinic acidcomprising more than one sinapic acid moiety;

tartaric acid, a tartaric acid derivative, an ester of tartaric acid, anester of a tartaric acid derivative, and

3-O-feruloylquinic acid, 4-O-feruloylquinic acid, 5-O-feruloylquinicacid, 3,4-diferuloylquinic acid, 3,5-diferuloylquinic acid,4,5-diferuloylquinic acid.

Caffeic Acid has the Structure:

Ferulic Acid has the Structure:

p-Coumaric Acid has the Structure:

Sinapic Acid has the Structure:

Quinic Acid has the Structure:

3-(3,4-dihydroxyphenyl)lactic Acid has the Structure:

Tartaric Acid has the Structure:

and can be in the D and L forms.

Examples of the esters of the various acids contemplated herein includethe ester of caffeic acid and quinic acid, which includesmonocaffeoylqunic acids (e.g., chlorogenic acid, neochlorogenic acid,and cryptochlorogenic acid), and dicaffeoylqunic acids (e.g.,1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid,1,5-dicaffeoylquinic acid, 3,4-dicaffeoylqunic acid,3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid), and saltsthereof:

with 4,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and3,4-dicaffeoylquinic acid being most prevalent in the compositionscontemplated herein and most prevalent in abundant in stevia, yerbamate, globe artichoke, and green coffee.

Examples of the esters of the various acids contemplated herein includethe ester of caffeic acid and tartaric acid, which includes cichoricacid having the structure:

which has two caffeic acid molecules inked to a tartaric acid core andcaftaric acid having the structure:

which has one caffeic acid molecule linked to a tartaric acid core.

Examples of the esters of the various acids contemplated herein includethe ester of caffeic acid and 3-(3,4-dihydroxyphenyl)lactic acidincluding, for example, rosmarinic acid, which has the structure:

Some or all of these steviol glycoside stabilizing compounds can beisolated from botanical sources, including but not limited to botanicalsources such as eucommoia ulmoides, honeysuckle, Nicotiana benthamiana,globe artichoke, cardoon, stevia Rebaudiana, monkfruit, coffee, coffeebeans, green coffee beans, tea, white tea, yellow tea, green tea, oolongtea, black tea, red tea, post-fermented tea, bamboo, heather, sunflower,blueberries, cranberries, bilberries, grouseberries, whortleberry,lingonberry, cowberry, huckleberry, grapes, chicory, eastern purpleconeflower, echinacea, Eastern pellitory-of-the-well, Upright pellitory,Lichwort, Greater celandine, Tetterwort, Nipplewort, Swallowwort,Bloodroot, Common nettle, Stinging nettle, Potato, Potato leaves,Eggplant, Aubergine, Tomato, Cherry tomato, Bitter apple, Thorn apple,Sweet potato, apple, Peach, Nectarine, Cherry, Sour cherry, Wild cherry,Apricot, Almond, Plum, Prune, Holly, Yerba mate, Mate, Guayusa, YauponHolly, Kuding, Guarana, Cocoa, Cocoa bean, Cacao, Cacao bean, Kola nut,Kola tree, Cola nut, Cola tree, Ostrich fem, Oriental ostrich fem,Fiddlehead fem, Shuttlecock fem, Oriental ostrich fem, Asian royal fem,Royal fem, Bracken, Brake, Common bracken, Eagle fem, Easternbrakenfern, Clove, Cinnamon, Indian bay leaf, Nutmeg, Bay laurel, Bayleaf, Basil, Great basil, Saint-Joseph's-wort, Thyme, Sage, Garden sage,Common sage, Culinary sage, Rosemary, Oregano, Wild marjoram, Marjoram,Sweet marjoram, Knotted marjoram, Pot marjoram, Dill, Anise, Star anise,Fennel, Florence fennel, Tarragon, Estragon, Mugwort, Licorice,Liquorice, Soy, Soybean, Soybean, Soya vean, Wheat, Common wheat, Rice,Canola, Broccoli, Cauliflower, Cabbage, Bok choy, Kale, Collard greens,Brussels sprouts, Kohlrabi, Winter's bark, Elderflower, Assa-Peixe,Greater burdock, Valerian, and Chamomile

The compositions comprising a SG and a steviol glycoside stabilizingcompound can comprise any suitable amount of steviol glycosidestabilizing compound, such as at least about 0.03 wt. % (e.g., at leastabout 0.1 wt. %, at least about 0.5 wt. %, at least about 0.6 wt. %, atleast about 1 wt. %, at least about 1.5 wt. %, at least about 2.5 wt. %,at least 3 wt. %, at least about 5 wt. %, at least about 6 wt. %, atleast about 10 wt. %, at least about 15 wt. %, at least about 20 wt. %,at least about 25 wt. %, at least about 30 wt. %, at least about 35 wt.%, or up to about 40 wt. %; about 0.03 wt. % to about 5 wt. %, about 0.1wt. % to about 10 wt. %, about 1 wt. % to about 4 wt. %, about 0.1 wt. %to about 5 wt. %, about or about 1 wt. % to about 5 wt. %) steviolglycoside stabilizing compound.

The compositions comprising a SG and a steviol glycoside stabilizingcompound can comprise a 1:0.3 to 1:3 (e.g., 1:1 to 1:1.5; or 1:1 to 1:2)ratio by weight of steviol glycoside to steviol glycoside stabilizingcompound.

Also contemplated herein are diastereomers and structural isomers of anyof the aforementioned acids. And because the aforementioned acids can beconsidered weak acids, they can each exist in at least one of theirconjugate acid form, conjugate base form (e.g., in their salt form), andmixed conjugate acid-conjugate base form, wherein a fraction (e.g., molefraction) of the compounds exist in the conjugate acid form and anotherfraction exist in the conjugate base form. The fraction of conjugateacid form to conjugate base of each acid will depend on various factors,including the pK_(a) of each compound and the pH of the composition.Examples of salts of any of the aforementioned acids include, but arenot limited to, quaternary ammonium, sodium, potassium, lithium,magnesium, and calcium salts of the one or more steviol glycosidestabilizing compounds, and the like.

An example of a method for making a composition comprising one or moresteviol glycoside stabilzing compounds, and salts thereof, comprises:

(a) contacting yerba mate biomass with an aqueous composition to obtainan initial extract;(b) removing solids from the initial extract to obtain a second initialextract;(c) adjusting the volume of the second initial extract with an aqueouscomposition to obtain an adjusted second initial extract;(d) chromatographing the adjusted second initial extract on an ionexchange chromatography stationary phase;(e) eluting the ion exchange chromatography stationary phase to obtain afirst eluent comprising a solvent;(f) removing the solvent to form a concentrate; and(g) at least one of decoloring and desaling the concentrate to at leastone of a filtrate and a retentate.

Another example of a method for making a composition comprising one ormore steviol glycoside stabilizing compounds, and salts thereof,comprises:

(a) contacting yerba mate biomass with an aqueous composition to obtainan initial extract;(b) removing solids from the initial extract to obtain a second initialextract;(c) adjusting the volume of the second initial extract with an aqueouscomposition to obtain an adjusted second initial extract;(d) chromatographing the adjusted initial extract on an ion exchangechromatography stationary phase;(e) eluting the ion exchange stationary phase to obtain a first eluentcomprising a solvent;(f) removing the solvent to form a concentrate;(g) at least one of decoloring and desalting the concentrate to obtainat least one of a filtrate and a retentate; and(h) drying the at least one of a filtrate and a retentate to obtain thecomposition comprising one or more steviol glycoside stabilizingcompounds, and salts thereof.

Step (a) of the methods described herein involve contacting yerba matebiomass with an aqueous composition to obtain an initial extractcomprising one or more steviol glycoside stabilizing compounds, andsalts thereof (e.g., quaternary ammonium, sodium, potassium, lithium,magnesium, and calcium salts).

The aqueous composition can comprise water and not contain anyco-solvents, such as organic solvents. But the aqueous composition cancomprise co-solvents, in addition to water. Suitable co-solvents includeorganic solvents, such as, (C₁-C₄)akanols and mixtures of(C₁-C₄)akanols. By “(C₁-C₄)alkanol” is meant an alcohol of the formula(C₁-C₄)alkyl-OH, wherein “alkyl” refers to straight chain and branchedalkyl groups having from 1 to 4 carbon atoms such as methyl, ethyl,n-propyl, n-butyl, isopropyl, iso-butyl, sec-butyl, and t-butyl, suchthat the resulting (C1-C₄)akanol is methanol, ethanol, n-propanol,n-butanol, isopropanol, iso-butanol, sec-butanol, and t-butanol. Theproportion of organic solvent, such as (C₁-C₄)akanol or mixtures of(C₁-C₄)akanols, can be any suitable proportion such that the aqueouscomposition can comprise up to about 30%, up to about 40%, up to about50% or up to about 60%, up to about 70%, up to about 80%, up to about90% or up to 100% by volume organic solvent the balance being water,except when the aqueous composition comprises 100% by volume organicsolvent; or from about 30% to about 100%, about 50% to about 100%, about60% to about 90%, about 30% to about 60%, about 40% to about 60%, about30% to about 50%, about 40% to about 50%, or about 50% by volume organicsolvent, the balance being water.

In some instances, the aqueous composition can be buffered with anysuitable buffering system, including, but not limited to, a phosphate,citrate, ascorbate, lactate, acetate, and the like. Buffers can be inthe range of 1-1000 mM of the anion. Alternatively, water acidified topH 5-6 with hydrochloric acid, sulfuric acid, nitric acid or the likecan be useful in the aqueous composition, with or without a co-solvent.Alternatively pure water made basic to pH 7-11 with hydroxide, such aswith sodium or potassium hydroxide, can be useful in the aqueouscomposition, with or without a co-solvent. In still other instances, itmay be suitable to add a suitable non-ionic solute that can help balancethe osmotic potential of the aqueous composition.

As used herein, the term “yerba mate biomass” generally refers to anyand all parts of the yerba mate plant, such as Ilex paraguariensis,including the yerba mate plant leaves, stalks, stems, tops, roots, andthe like. The yerba mate biomass can be in any suitable form includingin comminuted form resulting from, e.g., from chopping the yerba matebiomass prior to and/or during the contacting with the aqueouscomposition. For example, the yerba mate biomass can be comminuted in asuitable container and the aqueous composition can be added to thecomminuted yerba mate biomass, thus “contacting” the yerba mate biomass.The comminuted yerba mate biomass can then be optionally furthercomminuted within the suitable container. Or the yerba mate biomass canbe placed in a suitable container, to which the aqueous composition isadded, thus “contacting” the yerba mate biomass, and the resultingcomposition can be comminuted.

The yerba mate biomass can be stirred, sonicated or otherwise agitatedprior to and/or during the contacting to, among other things, maximizethe extraction of, among other of the acids described herein, the one ormore steviol glycoside stabilizing compounds, and salts thereof.

The initial extract can be carried through to step (c) as-is or bulksolids and or plant solids present, such as comminuted yerba mate plantleaves, stalks, tops, roots, and the like, can be removed in step (b) ofthe methods described herein. When step (b) is carried out, one obtainsa second initial extract.

Bulk solids can be removed by any suitable method, includingcentrifugation, skimming, or filtration. For example, the initialextract can be filtered using any suitable filtration method, includinggravity filtration or vacuum filtration through any suitable filter, solong as the filter does not substantially retain the one or more steviolglycoside stabilizing compounds, and salts thereof, including a paperfilter (e.g., low ash filter paper, such as Whatman 44 or 54 low ashfilter paper), a nylon filter, polyethersulfone filter, a glass fiberfilter, a pad of diatomaceous earth, and the like.

Step (c) of the methods described herein involves adjusting the volumeof the initial extract or second initial extract with a first aqueouscomposition or a second aqueous composition, respectively, to obtain anadjusted initial extract or adjusted second initial extract. The firstand second aqueous compositions can be different or the same. Theadjusted initial extract or adjusted second initial extract can befiltered at this point or can be carried through to step (d) as-is. Theinitial extract or the second initial extract can be filtered using anysuitable filtration method, including gravity filtration or vacuumfiltration through any suitable filter, so long as the filter does notsubstantially retain the one or more steviol glycoside stabilizingcompounds, and salts thereof, including a paper filter (e.g., low ashfilter paper, such as Whatman 44 or 54 low ash filter paper), a nylonfilter, polyethersulfone filter, a glass fiber filter, a pad ofdiatomaceous earth, and the like.

The volume of the initial extract or second initial extract can beadjusted with a sufficient amount of an aqueous composition (e.g.,water) to obtain an adjusted initial extract or adjusted second initialextract to, among other things, increase the binding of the one or moresteviol glycoside stabilizing compounds, and salts thereof, to the ionexchange chromatography column used in step (d) of the methods describedherein, relative to an unadjusted initial extract or an unadjustedsecond initial extract.

The volume of the initial extract or second initial extract can beadjusted to, among other things, adjust the amount of organic solvent,when present, in the initial extract or second initial extract. Thevolume of the initial extract or second initial extract can be adjustedsuch that the adjusted initial extract or adjusted second initialextract comprises less than about 60%, less than about 50%, less thanabout 40%, less than about 30%, less than about 20%, less than about10%, less than about 5%, less than about 1% or even about 0% by volumeorganic solvent, the balance being water; or from about 0% to about 40%,about 0% to about 30%, about 10% to about 40%, about 10% to about 30%,about 20% to about 40%, about 30% to about 40%, or about 35% by volumeorganic solvent, the balance being water.

Step (d) of the methods described herein involves chromatographing theadjusted initial extract or the second initial extract on an ionexchange stationary phase (e.g., a weak anion exchange stationaryphase). The chromatographing can be performed in any suitable fashion,including in batch mode or using a column. The chromatographing can beperformed with an aqueous composition (e.g., an aqueous compositioncomprising a (C₁-C₄)akanol) as eluent (e.g., an aqueous compositioncomprising from about 0% to about 40%, about 0% to about 30%, about 10%to about 40%, about 10% to about 30%, about 20% to about 40%, about 30%to about 40%, or about 35% by volume (C₁-C₄)akanol, the balance beingwater), leaving one or more steviol glycoside stabilizing compounds, andsalts thereof, adsorbed on the weak ion exchange chromatography column,while eluting other compounds including caffeine, rutin (also known asrutoside, quercetin-3-O-rutinoside, and sophorin)

and isomers thereof. Step (d) of the methods described herein candecrease the concentration of at least one of caffeine, rutin, and rutinisomers to a concentration of less than 1%, less than 0.5%, less than0.1%, less than 0.05%, less than 0.01% or less than 0.001% by mass. Theinstant disclosure therefore contemplates yerba mate extracts comprisingless than 0.1% of at least one of caffeine, rutin, and rutin isomers bymass. The instant disclosure also contemplates yerba mate extractscomprising less than 0.5% by mass of each one of caffeine, rutin, andrutin isomers and a less than about 1% by mass of caffeine, rutin, andrutin isomers combined. The instant disclosure also contemplates yerbamate extracts that are effectively free of at least one of caffeine,rutin, and rutin isomers (e.g., free of caffeine, free of rutin, free ofrutin isomers, and/or free of caffeine, rutin, and rutin isomers).

The ion exchange stationary phase is non-limiting and can be anysuitable ion exchange chromatography stationary phase. Examples ofsuitable ion exchange chromatography stationary phases includeANX-SEPHAROSE® fast flow resin, DEAE SEPHAROSE®, DEAE SEPHADEX® A25resin, Reite RAM2, Reite MG1P, AMBERLITE® (FPA 53; FPA 55; CG-50 Type I;IRC-50; IRC-50S; and IRP-64), DIAION WA10, and DOWEX® CCR-3.

The ion exchange chromatography stationary phase can optionally bepre-conditioned with an aqueous composition (e.g., an aqueouscomposition comprising a (C₁-C₄)alkanol), such as an aqueous compositioncomprising from about 0% to about 40%, about 0% to about 30%, about 10%to about 40%, about 10% to about 30%, about 20% to about 40%, about 30%to about 40%, or about 35% by volume (C₁-C₄)alkanol, the balance beingwater, prior to the chromatographing of the adjusted initial extract oradjusted second initial extract. For example, the weak ion exchangechromatography column can be pre-conditioned with about 2 or more bedvolumes (BV) at a flow rate of about 2 BV/h.

The pH of the weak ion exchange chromatography column can optionally beadjusted prior to the chromatographing of the adjusted initial extractor adjusted second initial extract. For example, the pH of the weak ionexchange chromatography column can be adjusted prior to thechromatographing with any suitable acid (e.g., hydrochloric acid) suchthat the pH of the weak ion exchange chromatography column (e.g., the pHof the resir/stationary phase) is a pH of less than about 10, about 9 orless, about 8 or less, about 7 or less, about 6 or less, about 5 orless, about 4 or less, about 3 or less; or a pH of about 2 to about 10,about 3 to about 8, about 5 to about 9, about 2 to about 6; about 3 toabout 4; or about 3 to about 6. The pH of the weak ion exchangechromatography column can be adjusted before or after the column isoptionally pre-conditioned with the aqueous composition comprising a(C₁-C₄) prior to the chromatographing of the adjusted initial extract oradjusted second initial extract.

After pre-conditioning and/or adjusting of the pH of the weak ionexchange chromatography column, the adjusted initial extract or adjustedsecond initial extract can be loaded onto the column at any suitablerate, such as at a rate of above 2 BV/h (bed volumes per hour). Afterloading the adjusted initial extract or adjusted second initial extract,the column can be washed with any suitable volume of an aqueouscomposition comprising a (C₁-C₄)alkanol (e.g., at least about 2 BV, atleast about 3 BV or at least about 4 BV of an aqueous compositioncomprising from about 10% to about 40%, about 10% to about 30%, about20% to about 40%, about 30% to about 40%, or about 35% by volume(C₁-C₄)alkanol, the balance being water) at any suitable rate, such asat a rate of about 2 BV/h. The volume of aqueous composition comprisinga (C₁-C₄)alkanol can be discarded, as it will contain, among otherthings, caffeine, rutin, and rutin isomers.

Step (e) of the methods described herein involves eluting the adsorbedone or more steviol glycoside stabilizing compounds, and salts thereof,from the weak ion exchange chromatography column to obtain a firsteluent comprising one or more steviol glycoside stabilizing compounds,and salts thereof. The eluting is performed under any conditionssuitable to elute the one or more steviol glycoside stabilizingcompounds, and salts thereof from the column.

An example of suitable conditions to elute the one or more steviolglycoside stabilizing compounds, and salts thereof from the columninclude eluting the column with any suitable volume of a solutioncomprising a salt (e.g., sodium chloride, potassium chloride, ammoniumchloride, sodium sulfate, potassium sulfate, sodium phosphate, potassiumphosphate, and the like). Examples of solutions comprising a saltinclude solutions comprising at least one salt (e.g., about 5 wt. % toabout 25 wt. %, about 15 wt. % to about 20 wt. % or about 5 wt. % toabout 10 wt. % of a salt) dissolved in an aqueous composition comprisinga (C₁-C₄)alkanol (e.g., at least about 2 BV, at least about 3 BV or atleast about 4 BV of an aqueous composition comprising from about 10% toabout 60%, about 20% to about 50%, about 30% to about 55%, about 40% toabout 60%, or about 50% by volume (C₁-C₄)alkanol).

Another example of suitable conditions to elute the one or more steviolglycoside stabilizing compounds, and salts thereof from the columninclude eluting the column with any suitable volume of a solutioncomprising an acid (e.g., hydrochloric acid, sulfuric acid, phosphoricacid, acetic acid, formic acid, and the like). Examples of solutionscomprising an acid include solutions comprising hydrochloric acid andthe like and optionally acids solutions comprising an aqueouscomposition comprising from about 10% to about 60%, about 20% to about50%, about 30% to about 55%, about 40% to about 60%, or about 50% byvolume (C₁-C₄)alkanol).

The first eluent comprising the one or more steviol glycosidestabilizing compounds, and salts thereof, collected from the elutingstep is collected and can be subsequently concentrated by removingsolvent (e.g., to remove water and (C₁-C₄)alkanol) by any suitable meansto provide a concentrate comprising the one or more steviol glycosidestabilzing compounds, and salts thereof. The solvent removal can beaccomplished under an inert atmosphere (e.g., under a nitrogen gasatmosphere). While not wishing to be bound by any specific theory, it isbelieved that performing the solvent removal under an inert atmospherecan reduce the formation of highly colored polymeric substances thateither natively exist in the yerba mate biomass or form at one or moreof the steps described herein.

The first eluent comprising the one or more steviol glycosidestabilizing compounds, and salts thereof comprises a solvent. Thesolvent can be removed in a step (f) to dryness or it can be removed toa point where a volume of an aqueous composition comprising a(C₁-C₄)alkanol remains as a solvent (e.g., about 50%, about 40%, about30% about 20%, about 10% or about 5% of an original, total volume of theeluent) to form a concentrate, though the ratio of components that makeup the aqueous composition comprising a (C₁-C₄)alkanol may or may not bedifferent from the ratio of components that made up the aqueouscomposition comprising a (C₁-C₄)alkanol that was used to elute theadsorbed one or more steviol glycoside stabilizing compounds, and saltsthereof. Alternatively, the solvent in the eluent comprising the one ormore steviol glycoside stabilizing compounds, and salts thereof, can beremoved to a point where a volume of an aqueous composition comprising a(C₁-C₄)alkanol remains, wherein the aqueous composition comprising a(C₁-C₄)alkanol comprises less than about 10%, less than about 5%, lessthan about 2% or less than about 1% by volume (C₁-C₄)alkanol.

Suitable conditions for removing solvent from the eluent comprising,among other of the acids described herein, the one or more steviolglycoside stabilizing compounds, and salts thereof, to form aconcentrate comprising, among other of the acids described herein, theone or more steviol glycoside stabilizing compounds, and salts thereofinclude blowing an inert gas (e.g., nitrogen gas) over the surface ofthe eluent. The eluent can be heated while blowing the nitrogen gas orit can be at room temperature (e.g., 25° C.). Other conditions forremoving the solvent in the eluent include applying a vacuum to thecontainer containing the eluent. The vacuum can be applied with theeluent at room temperature or while heating the container. Yet otherconditions for removing solvent in the eluent include passing the eluentthrough a wiped film evaporator or an agitated thin film evaporator.

The pH of the concentrate can be adjusted at this point to obtain apH-adjusted concentrate, though adjusting the pH at this point isoptional. For example, the pH of the concentrate can be adjusted to a pHwhere, among other of the acids described herein, the one or moresteviol glycoside stabilizing compounds, and salts thereof are protectedfrom degradation. Suitable pHs include pHs of less than about 6, lessthan about 5, less than about 4, less than about 3 or less than about 2;such as a pH of from about 2 to about 6, about 2 to about 5, about 2 toabout 4, about 3 to about 5 or a pH of about 3.5. The pH of theconcentrate can be adjusted by using any suitable acid or base. When anacid is used, the acid can be hydrochloric acid and the like.

The concentrate or the pH-adjusted concentrate can be taken on as-is inthe methods described herein or the removing step (f) or they can befiltered. The concentrate or the pH-adjusted concentrate can be filteredusing any suitable filter (e.g., low ash filter paper, such as Whatman44 or 54 low ash filter paper), a nylon filter, a polyethersulfonefilter, a glass fiber filter, a pad of diatomaceous earth, and the like.In some instances, the pH-adjusted concentrate can be filtered through apolymeric membrane, such as a polyethersulfone (PES) filter having,e.g., 0.2 μm pore size, or a pleated (flat membrane, vacuum filtration)or a pleated PES membrane, depending on the volume of the concentrate orthe pH-adjusted concentrate.

The concentrate comprising one or more steviol glycoside stabilizingcompounds, and salts thereof, whether it is pH-adjusted, filtered orboth pH-adjusted and filtered, can be taken directly to drying step (h)or can be submitted for desalting/decoloring in step (g) (in eitherorder, including desalting, followed by decoloring; decoloring, followedby desalting; decoloring, but not desalting; or desalting, but notdecoloring) of a concentrate that can be highly colored. Thedesalting/decoloring can be accomplished under an inert atmosphere(e.g., under a nitrogen gas atmosphere). While not wishing to be boundby any specific theory, it is believed that performing the one or moresteps under an inert atmosphere can reduce the formation of highlycolored polymeric substances that either natively exist in the yerbamate biomass or form at one or more of the steps described herein.

The concentrate, whether it is pH-adjusted, filtered or both pH-adjustedand filtered, can be decolored by any suitable means, includingultrafiltration (e.g., filtering through a molecular weight cutoffmembrane, size-exclusion chromatography or gel permeation). One obtainsa filtrate from decoloring. Ultrafiltration accomplishes, among otherthings, decoloration of a concentrate that can be highly colored. Whilenot wishing to be bound by any specific theory, it is believed thatultrafiltration removes highly colored polymeric substances that eithernatively exist in the yerba mate biomass or form at one or more of thesteps described herein.

The filtrate from decoloring can be taken on to drying step (h) or itcan be desalted in step (g). Alternatively, the concentrate, whether itis pH-adjusted, filtered or both pH-adjusted and filtered, can bedesalted without first decoloring. Regardless, the desalting can beaccomplished using a nanofiltration membrane and a hydrophobic resin.Those of skill in the art would recognize that when one uses ananofiltration membrane and a hydrophobic resin one discards thepermeate and keeps the retentate. In one example, desalting can beaccomplished using a hydrophobic resin (e.g., a porous polydivinylbenzene/ethylvinylbenzene matrix, such as SEPABEADS™ SP70), whereone would load a pH-adjusted concentrate (e.g., an acidifiedconcentrate, with a pH of less than about 2) comprising less than about20% by volume (C₁-C₄)alkanol. The resin is then washed with dilutealcohol (e.g., less than about 10% by volume (C₁-C₄)alkanol, the restbeing water having a pH of less than about 2) and then eluted with anaqueous composition comprising about 70% by volume (C₁-C₄)alkanol inwater to obtain a desalted second eluent comprising one or more steviolglycoside stabilizing compounds, and salts thereof.

If desalting precedes decoloring in step (g), the solvent in thepermeate from the desalting step can be removed to a point where avolume of an aqueous composition comprising a (C₁-C₄)alkanol remains asa solvent (e.g., about 50%, about 40%, about 30% about 20%, about 10% orabout 5% of an original, total volume of the eluent) to form a firstdesalted concentrate. Alternatively, the solvent in the permeate fromthe desalting can be removed, to give a second desalted concentrate, toa point where a volume of an aqueous composition comprising a(C₁-C₄)alkanol remains, wherein the aqueous composition comprising a(C₁-C₄)alkanol comprises less than about 10%, less than about 5%, lessthan about 2% or less than about 1% by volume (C₁-C₄)alkanol. The firstdesalted concentrate can also have the attributes of the second desaltedconcentrate, such that the first desalted concentrate also has less thanabout 10%, less than about 5%, less than about 2% or less than about 1%by volume (C₁-C₄)alkanol.

Suitable conditions for removing solvent from the permeate comprisingone or more steviol glycoside stabilizing compounds, and salts thereof,to form a first/second desalted concentrate comprising one or moresteviol glycoside stabilizing compounds, and salts thereof includeblowing an inert gas (e.g., nitrogen gas) over the surface of theeluent. The permeate can be heated while blowing the nitrogen gas or itcan be at room temperature (e.g., 25° C.). Other conditions for removingthe solvent in the eluent include applying a vacuum to the containercontaining the permeate. The vacuum can be applied with the permeate atroom temperature or while heating the container. Yet other conditionsfor removing solvent in the permeate include passing the permeatethrough a wiped film evaporator or an agitated thin film evaporator.

In another example, the concentrate comprising one or more steviolglycoside stabilizing compounds, and salts thereof can be filteredthrough filter paper to obtain a first filtrate, the first filtrate isultrafiltered to obtain a second filtrate, and the second filtrate isnanofiltered using a nanofiltration membrane to obtain a first retentateor the second filtrate is eluted through a hydrophobic resin to obtain adesalted second eluent. In another example, the concentrate comprisingone or more steviol glycoside stabilizing compounds, and salts thereofcan be filtered through filter paper to obtain a first filtrate, thefirst filtrate is nanofiltered using a nanofiltration membrane to obtaina third retentate or the first filtrate is eluted through a hydrophobicresin to obtain a desalted second eluent, and the third retentate or thedesalted second eluent is ultrafiltered to obtain a third filtrate.

As mentioned herein, the eluent comprising one or more steviol glycosidestabilizing compounds, and salts thereof, can be concentrated to drynessor it can be concentrated to a point where a volume of an aqueouscomposition comprising a (C₁-C₄)alkanol remains. If the eluent isconcentrated to dryness, the dry material can be reconstituted using,for example, an aqueous composition comprising a (C₁-C₄)alkanol. Thereconstituted material can then be filtered as described herein, toamong other things, at least one of desalt and decolor.

The methods described herein can include step (h) that involves dryingfirst retentate, desalted second eluent or the third filtrate to obtainthe composition comprising one or more steviol glycoside stabilizingcompounds, and salts thereof. The first retentate, desalted secondeluent or the third filtrate can be dried in any suitable manner,including by lyophilization or spray drying.

FIG. 1 is a flow diagram of a method 100 for making a compositioncomprising one or more steviol glycoside stabilizing compounds, andsalts thereof. In operation 102, yerba mate biomass is contacted with anaqueous composition containing 50% ethanol/water in a suitable container(e.g., a glass jar) for 1 h (300 g yerba mate biomass into 1.5 Lsolvent) to obtain an initial extract. In operation 104, the initialextract is filtered using, for example, a ceramic Büchner funnel withWhatman 54 low ash filter paper into glass 4 L side arm flask. Inoperation 106, the volume of the filtered initial extract is adjustedwith an aqueous composition, in this case water, to obtain an adjustedfiltered initial extract containing a lower proportion of ethanol, inthis case 35% by volume ethanol. In operation 108, the adjusted filteredinitial extract can be re-filtered using, for example, a ceramic Büchnerfunnel with Whatman 44 low ash filter paper into glass 4 L side armflask. In operation 110, the adjusted filtered initial extract ischromatographed on an ion exchange chromatography stationary phase. Forexample, AMBERLITE® FPA 53 resin is packed in glass column. The resin ispreconditioned with 35% ethanol (2 BV at 2 BV/h). The adjusted filteredinitial extract is loaded is loaded (2 BV/h) onto the resin, discardingthe loading permeate. The resin is washed with 35% ethanol (4 BV at 2BV/h) discarding the washing permeate. The one or more steviol glycosidestabilizing compounds, and salts thereof are eluted with 50%ethanol/water, 10% FCC sodium chloride (4 BV, 0.5 BV/h) and the permeateis kept. The column/resin can optionally be regenerated with water (4BV, 2 BV/h). In operation 112, the eluent/permeate is concentrated toform a concentrate. In this case, nitrogen gas was blown over the top ofthe eluent/permeate for 2 days, until volume the volume is approximatelyone third of the initial volume of eluent/permeate and/or ethanol isless than 1% in the eluent/permeate, thereby obtaining a concentrate. Inoperation 114, the concentrate is acidified to a pH of approximately 3.5and then filtered through a Whatman 44 filter paper on a Büchner funnelfollowed by 0.2 μm polyether sulfone (PES) filter. In operation 116, thefiltered concentrate is decolored using a molecular weight cutoffmembrane (MWCO; e.g., a MWCO membrane that removes materials having amolecular weight of greater than 10 kDA, such as a 3 kDa TURBOCLEAN®NP010 or Synder VT-2B or a 1 kDa Synder XT-2B) to, among other things,decolor the filtered concentrate and obtain a permeate. In operation118, the permeate is filtered through a nanofiltration membrane (e.g.,TRISEP XN45 membrane) and the retentate is subsequently dried inoperation 120 to obtain the composition comprising one or more steviolglycoside stabilizing compounds, and salts thereof.

FIG. 2 is a flow diagram of a method 200 for making a compositioncomprising one or more steviol glycoside stabilizing compounds, andsalts thereof. In operation 202, yerba mate biomass is contacted with anaqueous composition containing 50% ethanol/water in a suitable container(e.g., a glass jar) for 1 h (300 g yerba mate biomass into 1.5 Lsolvent) to obtain an initial extract. In operation 204, the initialextract is filtered using, for example, a ceramic Büchner funnel withWhatman 54 low ash filter paper into glass 4 L side arm flask. Inoperation 206, the volume of the filtered initial extract is adjustedwith an aqueous composition, in this case water, to obtain an adjustedfiltered initial extract containing a lower proportion of ethanol, inthis case 35% by volume ethanol. In operation 208, the adjusted filteredinitial extract can be re-filtered using, for example, a ceramic Büchnerfunnel with Whatman 44 low ash filter paper into glass 4 L side armflask. In operation 210, the adjusted filtered initial extract ischromatographed on an ion exchange chromatography stationary phase. Forexample, AMBERLITE® FPA 53 resin is packed in glass column. The resin ispreconditioned with 35% ethanol (2 BV at 2 BV/h). The adjusted filteredinitial extract is loaded is loaded (2 BV/h) onto the resin, discardingthe loading permeate. The resin is washed with 35% ethanol (4 BV at 2BV/h) discarding the washing permeate. The one or more steviol glycosidestabilizing compounds, and salts thereof are eluted with 50%ethanol/water, 10% FCC sodium chloride (4 BV, 0.5 BV/h) and the permeateis kept. The column/resin can optionally be regenerated with water (4BV, 2 BV/h). In operation 212, the eluent/permeate is concentrated toform a concentrate, where the volume is approximately one third of theinitial volume of eluent/permeate and/or ethanol is less than 1% in theeluent/permeate, thereby obtaining a concentrate. In operation 214, theconcentrate is acidified to a pH of approximately 1 and then filteredthrough a Whatman 44 filter paper on a Büchner funnel followed by 0.2 μmpolyether sulfone (PES) filter. In operation 218, the concentrate isdesalted using a hydrophobic resin (e.g., a porous polydivinylbenzene/ethylvinylbenzene matrix, such as SEPABEADS™ SP70) andthe solvent in the retentate is removed in operation 217. In operation216, the desalted concentrate is decolored using a molecular weightcutoff membrane (MWCO; e.g., a MWCO membrane that removes materialshaving a molecular weight of greater than 10 kDA, such as a 3 kDaTURBOCLEAN® NP010) to, among other things, decolor the filteredconcentrate and obtain a permeate, which is subsequently dried inoperation 220 to obtain the composition comprising one or more steviolglycoside stabilizing compounds, and salts thereof.

Another example of a method for making a composition comprising one ormore steviol glycoside stabilizing compounds, and salts thereof, themethod comprising

(i) contacting yerba mate biomass with an aqueous composition to obtainan initial extract;(ii) removing solids from the initial extract to obtain a second initialextract;(iii) contacting the second initial extract with acidified ethyl acetateto obtain an acidic ethyl acetate extract;(iv) neutralizing the acidic ethyl acetate extract to obtain neutralizedethyl acetate and an aqueous extract;(v) decoloring the aqueous extract to obtain a decolored aqueousextract; and(vi) drying the decolored aqueous extract to obtain the compositioncomprising one or more steviol glycoside stabilizing compounds, andsalts thereof.

Steps (i), (ii), and (vi) are performed as described herein for steps(a), (b), and (h). Step (v) is analogous to filtering step (g), exceptthat step (v) involves only decoloring processes, such asultrafiltration, which includes filtering through a molecular weightcutoff membrane, size-exclusion chromatography, and gel permeation, asdiscussed herein. Accordingly, the disclosure with regard to steps (a),(b), (g), and (h) apples to steps (i), (ii), (v), and (vi).

Step (i) of the methods described herein involve contacting yerba matebiomass with an aqueous composition to obtain an initial extractcomprising one or more steviol glycoside stabilizing compounds, andsalts thereof.

The aqueous composition can comprise water and not contain anyco-solvents, such as organic solvents. But the aqueous composition cancomprise co-solvents, in addition to water. Suitable co-solvents includeorganic solvents, such as, (C₁-C₄)alkanols and mixtures of(C₁-C₄)alkanols. The proportion of organic solvent, such as(C₁-C₄)alkanol or mixtures of (C₁-C₄)alkanols, can be any suitableproportion such that the aqueous composition can comprise up to about30%, up to about 40%, up to about 50% or up to about 60% by volumeorganic solvent, the balance being water; or from about 30% to about60%, about 40% to about 60%, about 30% to about 50%, about 40% to about50%, or about 50% by volume organic solvent, the balance being water.

In some instances, the aqueous composition can be buffered with anysuitable buffering system, including, but not limited to, a phosphate,citrate, ascorbate, lactate, acetate, and the like. Buffers can be inthe range of 1-1000 mM of the anion. Alternatively, water acidified topH 5-6 with hydrochloric acid, sulfuric acid, nitric acid or the likecan be useful in the aqueous composition, with or without a co-solvent.Alternatively, pure water made basic to pH 7-11 with hydroxide, such assodium or potassium hydroxide can be useful in the aqueous composition,with or without a co-solvent. In still other instances, it may besuitable to add a suitable non-ionic solute that can help balance theosmotic potential of the aqueous composition.

The yerba mate biomass can be stirred, sonicated or otherwise agitatedprior to and/or during the contacting of step (i) to, among otherthings, maximize the extraction of one or more steviol glycosidestabilizing compounds, and salts thereof.

The initial extract can be carried through to step (iii) as-is or bulksolids and or plant solids present, such as comminuted yerba mate plantleaves, stalks, tops, roots, and the like, can be removed in step (ii)of the methods described herein. When step (ii) is carried out, oneobtains a second initial extract.

Bulk solids can be removed by any suitable method, includingcentrifugation, skimming, or filtration. For example, the initialextract can be filtered using any suitable filtration method, includinggravity filtration or vacuum filtration through any suitable filter, solong as the filter does not substantially retain the one or more steviolglycoside stabilizing compounds, and salts thereof, including a paperfilter (e.g., low ash filter paper, such as Whatman 44 or 54 low ashfilter paper), a nylon filter, polyethersulfone filter, a glass fiberfilter, a pad of diatomaceous earth, and the like.

Prior to carrying out step (iii) one can optionally adjust the pH of theinitial or second initial extract with a suitable acid. (e.g.,hydrochloric acid and the like) or suitable base (e.g., sodiumhydroxide) to a pH of between about 4 and about 7. The pH-adjustedinitial or second initial extract is then extracted with ethyl acetatethat has not been pre-acidified as described herein. While not wishingto be bound by any specific theory, it is believed that when the pH ofthe initial or second initial extract is adjusted to between about 4 andabout 7, it is possible to extract certain impurities into the ethylacetate, while keeping the one or more steviol glycoside stabilizingcompounds in the aqueous layer.

Step (iii) of the methods described herein involves contacting the firstor second initial extract with acidified ethyl acetate to obtain anacidic ethyl acetate extract. The acidified ethyl acetate can beprepared in any suitable manner, including by adding any suitable acid,including hydrochloric acid, sulfuric acid, and glacial acetic acid(e.g., 0.01-1% vol/vol). The acidic ethyl acetate extract is washed withwater (e.g., three times, with 1:1 vol/vol water). Under theseconditions, the one or more steviol glycoside stabilizing compounds, andsalts thereof, will substantially be in their conjugate acid form andwill reside substantially in the acidic ethyl acetate layer that formswhen the acidic ethyl acetate extract is washed with water. The waterlayers are discarded and the acidic ethyl acetate extract is carried onto step (iv).

Step (iii) of the methods described herein can be carried out in othersuitable ways, including by using ethyl acetate that has not beenpre-acidified as described herein (e.g., by pre-washing with glacialacetic acid), but instead by adjusting the pH of the initial or secondinitial extract with a suitable acid. (e.g., hydrochloric acid and thelike), then extracting the pH-adjusted initial or second initial extractwith ethyl acetate that has not been pre-acidified. Regardless of theacid used to adjust the pH of the initial extract or the second initialextract, the pH of the initial extract or the second initial extract isadjusted to about 4 or less, 3 or less, about 2 or less, or about 1 orless. The water layers are discarded and the acidic ethyl acetateextract that results is carried on to step (iv).

Step (iv) of the methods described herein involves neutralizing theacidic ethyl acetate extract to obtain neutralized ethyl acetate and anaqueous extract. This is accomplished in any suitable way, includingwashing the acidic ethyl acetate extract with water (e.g., three times,with 1:1 vol/vol water) comprising a suitable base, such as sodiumhydroxide, potassium hydroxide, and the like, and combinations thereof.Under these conditions, the one or more steviol glycoside stabilizingcompounds, and salts thereof, will substantially be in their conjugatebase form and will substantially reside in the water layer that formswhen the acidic ethyl acetate extract is washed with water comprising asuitable base.

In an alternative, optional step to step (iv), step (iv-a), the acidicethyl acetate extract that results from step (iii) can be optionallyremoved, even removed to dryness. Any solid that remains can either bereconstituted with pH neutral water (e.g., deionized water) and the pHof the water can then be adjusted to about 3 to about 7; or the solidthat remains can be reconstituted with water having a pH of about 3 toabout 7.

The aqueous extract comprising the one or more steviol glycosidestabilizing compounds, and salts thereof, whether they emanate from step(iv) or step (iv-a), can then be submitted for step (v) to accomplish,among other things, decoloring of aqueous extract, which can be highlycolored. Decoloring can be accomplished by any suitable means, includingultrafiltration (e.g., filtering through a molecular weight cutoffmembrane, size-exclusion chromatography, or gel permeation). One obtainsa filtrate from decoloring. Ultrafiltration accomplishes, among otherthings, decoloration of a concentrate that can be highly colored. Whilenot wishing to be bound by any specific theory, it is believed thatultrafiltration removes highly colored polymeric substances that eithernatively exist in the yerba mate biomass or form at one or more of thesteps described herein.

Another example of modifications to the method described hereincomprising steps (i)-(vi) (including the alternative, optional step(iv-a) includes a method for making a composition comprising one or moresteviol glycoside stabilizing compounds, and salts thereof, the methodcomprising contacting yerba mate biomass with an aqueous composition toobtain an initial extract;

removing solids from the initial extract to obtain a second initialextract; adjusting the pH of the second initial extract to a pH of fromabout 4 to about 7 to obtain a first pH-adjusted second initial extract;contacting the first pH-adjusted second initial extract with ethylacetate to obtain a first ethyl acetate extract and a second aqueousextract;adjusting the pH of the second aqueous extract to a pH of less than 2 toobtain a pH-adjusted second aqueous extract;contacting the pH-adjusted second aqueous extract with ethyl acetate toobtain a second ethyl acetate extract;removing the ethyl acetate from the second ethyl acetate extract toobtain a purified composition;reconstituting the crude composition with water to obtain a thirdaqueous extract; anddecoloring the third aqueous extract to obtain a decolored aqueousextract. The “purified composition” will comprise the compounds ofinterest (e.g., the one or more steviol glycoside stabilizing compounds,and salts thereof) and is purified relative to at least the initialextract and the second initial extract, in that the “purifiedcomposition” will not contain certain impurities in the initial extractand the second initial extract, but does contain highly coloredpolymeric substances that either natively exist in the yerba matebiomass or form at one or more of the steps described herein and thatare removed in the decoloring step.

Yet another example of modifications to the method described hereincomprising steps (i)-(vi) (including the alternative, optional step(iv-a) includes a method for making a composition comprising one or moresteviol glycoside stabilizing compounds, and salts thereof, the methodcomprising contacting yerba mate biomass with an aqueous composition toobtain an initial extract;

removing solids from the initial extract to obtain a second initialextract;adjusting the pH of the second initial extract to a pH of less thanabout 2 to obtain a second pH-adjusted second initial extract;contacting the second pH-adjusted second initial extract with ethylacetate to obtain a third ethyl acetate extract;neutralizing the third ethyl acetate extract to obtain a firstneutralized ethyl acetate extract and a third aqueous extract; anddecoloring the third aqueous extract to obtain a decolored aqueousextract

The methods described herein can include step (vi) that involves dryingthe decolored aqueous extract to obtain the composition comprising oneor more steviol glycoside stabilizing compounds, and salts thereof. Thefirst or second retentates or the third filtrate can be dried in anysuitable manner, including by lyophilization or spray drying.

FIG. 3 is a flow diagram of a method 300 for making a compositioncomprising one or more steviol glycoside stabilizing compounds, andsalts thereof. In operation 302, yerba mate biomass is contacted with anaqueous composition containing 50% ethanol/water in a suitable container(e.g., a glass jar) for 1 h (300 g yerba mate biomass into 1.5 Lsolvent) to obtain an initial extract. In operation 304, the initialextract is filtered using, for example, a ceramic Büchner funnel withWhatman 54 low ash filter paper into glass 4 L side arm flask to, amongother things, remove solids from, e.g., the yerba mate biomass. Thefiltrate from operation 304 is extracted in operation 306 with acidifiedethyl acetate extraction. Following extraction of the one or moresteviol glycoside stabilizing compounds into the acidified ethylacetate, the acidified ethyl acetate is washed with water comprising asuitable base, such as sodium hydroxide, potassium hydroxide, and thelike, in operation 308 to obtain neutralized ethyl acetate and anaqueous extract Under these conditions, the one or more steviolglycoside stabilizing compounds will substantially be in their conjugatebase form and will substantially reside in the water layer that formswhen the acidic ethyl acetate extract is washed with water comprising asuitable base. In operation 310 the water layer is filtered to obtain afiltrate. In operation 316, the filtrate is decolored using a 3 kDamolecular weight cutoff membrane (TURBOCLEAN® NP010; six diafiltrations)to, among other things, decolor the aqueous extract, thereby obtaining adecolored aqueous extract. In operation 320, the decolored aqueousextract is dried to obtain the composition comprising one or moresteviol glycoside stabilizing compounds, and salts thereof.

FIG. 4 is a flow diagram of a method 400 for making a compositioncomprising one or more steviol glycoside stabilizing compounds, andsalts thereof. In operation 402, yerba mate biomass is contacted with anaqueous composition containing 50% ethanol/water in a suitable container(e.g., a glass jar) for 1 h (300 g yerba mate biomass into 1.5 Lsolvent) to obtain an initial extract. In operation 404, the initialextract is filtered using, for example, a ceramic Büchner funnel withWhatman 54 low ash filter paper into glass 4 L side arm flask to, amongother things, remove solids from, e.g., the yerba mate biomass. Thefiltrate from operation 404 is pH-adjusted to from about 4 to about 7and the filtrate is extracted in operation 408 with ethyl acetate, whilethe compounds of interest remain in the aqueous layer. In operation 406,the pH of the aqueous layer is adjusted to less than 2 and the aqueouslayer is extracted with ethyl acetate. Following extraction of the oneor more steviol glycoside stabilizing compounds into the ethyl acetate,the ethyl acetate is removed to dryness in operation 407 to obtain asolid. The solid is reconstituted with water and the pH of the water isadjusted to from about 3 to about 7. Under these conditions, the one ormore steviol glycoside stabilizing compounds will substantially be intheir conjugate base form and will dissolve in the water. In operation410 the water layer is filtered to obtain a filtrate. In operation 416,the filtrate is decolored using a 3 kDa molecular weight cutoff membrane(TURBOCLEAN® NP010; six diafiltrations) to, among other things, decolorthe aqueous extract, thereby obtaining a decolored aqueous extract. Inoperation 420, the decolored aqueous extract is dried to obtain thecomposition comprising one or more steviol glycoside stabilizingcompounds, and salts thereof.

The composition comprising the one or more steviol glycoside stabilizingcompounds, and salts thereof prepared according to the methods describedherein can be incorporated into any ingestible composition, includinginto beverages and food products.

For example, the ingestible composition can be a comestible compositionor noncomestible composition. By “comestible composition”, it is meantany composition that can be consumed as food by humans or animals,including solids, gel, paste, foamy material, semi-solids, liquids, ormixtures thereof. By “noncomestible composition”, it is meant anycomposition that is intended to be consumed or used by humans or animalsnot as food, including solids, gel, paste, foamy material, semi-solids,liquids, or mixtures thereof. The noncomestible composition includes,but is not limited to medical compositions, which refers to anoncomestible composition intended to be used by humans or animals fortherapeutic purposes. By “animal”, it includes any non-human animal,such as, for example, farm animals and pets.

The composition comprising the one or more steviol glycoside stabilizingcompounds, and salts thereof prepared according to the methods describedherein can be added to a noncomestible composition or non-edibleproduct, such as supplements, nutraceuticals, functional food products(e.g., any fresh or processed food claimed to have a health-promotingand/or disease-preventing properties beyond the basic nutritionalfunction of supplying nutrients), pharmaceutical and over the countermedications, oral care products such as dentifrices and mouthwashes,cosmetic products such as lip balms and other personal care products.

In general, over the counter (OTC) product and oral hygiene productgenerally refer to product for household and/or personal use which maybe sold without a prescription and/or without a visit to a medicalprofessional. Examples of the OTC products include, but are not limitedto Vitamins and dietary supplements; Topical analgesics and/oranaesthetic; Cough, cold and allergy remedies; Antihistamines and/orallergy remedies; and combinations thereof. Vitamins and dietarysupplements include, but are not limited to vitamins, dietarysupplements, tonics/bottled nutritive drinks, child-specific vitamins,dietary supplements, any other products of or relating to or providingnutrition, and combinations thereof. Topical analgesics and/oranaesthetic include any topical creams/ointments/gels used to alleviatesuperficial or deep-seated aches and pains, e.g. muscle pain; teethinggel; patches with analgesic ingredient; and combinations thereof. Cough,cold and allergy remedies include, but are not limited to decongestants,cough remedies, pharyngeal preparations, medicated confectionery,antihistamines and child-specific cough, cold and allergy remedies; andcombination products. Antihistamines and/or allergy remedies include,but are not limited to any systemic treatments for hay fever, nasalallergies, insect bites and stings. Examples of oral hygiene productsinclude, but are not limited to mouth cleaning strips, toothpaste,toothbrushes, mouthwashes/dental rinses, denture care, mouth freshenersat-home teeth whiteners and dental floss.

The composition comprising one or more steviol glycoside stabilizingcompounds, and salts thereof prepared according to the methods describedherein can be added to food or beverage products or formulations.Examples of food and beverage products or formulations include, but arenot limited to coatings, frostings, or glazes for comestible products orany entity included in the Soup category, the Dried Processed Foodcategory, the Beverage category, the Ready Meal category, the Canned orPreserved Food category, the Frozen Processed Food category, the ChilledProcessed Food category, the Snack Food category, the Baked Goodscategory, the Confectionary category, the Dairy Product category, theIce Cream category, the Meal Replacement category, the Pasta and Noodlecategory, and the Sauces, Dressings, Condiments category, the Baby Foodcategory, and/or the Spreads category.

In general, the Soup category refers to canned/preserved, dehydrated,instant, chilled, UHT and frozen soup. For the purpose of thisdefinition soup(s) means a food prepared from meat, poultry, fish,vegetables, grains, fruit and other ingredients, cooked in a liquidwhich may include visible pieces of some or all of these ingredients. Itmay be clear (as a broth) or thick (as a chowder), smooth, pureed orchunky, ready to serve, semi condensed or condensed and may be servedhot or cold, as a first course or as the main course of a meal or as abetween meal snack (sipped like a beverage). Soup may be used as aningredient for preparing other meal components and may range from broths(consommé) to sauces (cream or cheese based soups).

“Dehydrated and Culinary Food Category” usually means: (i) Cooking aidproducts such as: powders, granules, pastes, concentrated liquidproducts, including concentrated bouillon, bouillon and bouillon likeproducts in pressed cubes, tablets or powder or granulated form, whichare sold separately as a finished product or as an ingredient within aproduct, sauces and recipe mixes (regardless of technology); (ii) Mealsolutions products such as: dehydrated and freeze dried soups, includingdehydrated soup mixes, dehydrated instant soups, dehydrated ready tocook soups, dehydrated or ambient preparations of ready-made dishes,meals and single serve entrees including pasta, potato and rice dishes;and (iii) Meal embellishment products such as: condiments, marinades,salad dressings, salad toppings, dips, breading, batter mixes, shelfstable spreads, barbecue sauces, liquid recipe mixes, concentrates,sauces or sauce mixes, including recipe mixes for salad, sold as afinished product or as an ingredient within a product, whetherdehydrated, liquid or frozen.

The Beverage category means beverages, beverage mixes and concentrates,including but not limited to, carbonated and non-carbonated beverages,alcoholic and nonalcoholic beverages, ready to drink beverages, liquidconcentrate formulations for preparing beverages such as sodas, and drypowdered beverage precursor mixes. The Beverage category also includethe alcoholic drinks, the soft drinks, sports drinks, isotonicbeverages, and hot drinks. The alcoholic drinks include, but are notlimited to beer, cider/perry, FABs, wine, and spirits. The soft drinksinclude, but are not limited to carbonates, such as colas and non-colacarbonates; fruit juice, such as juice, nectars, juice drinks and fruitflavored drinks; bottled water, which includes sparking water, springwater and purified/table water; functional drinks, which can becarbonated or still and include sport, energy or elixir drinks;concentrates, such as liquid and powder concentrates in ready to drinkmeasure. The hot drinks include, but are not limited to coffee, such asfresh (e.g., brewed), instant, combined coffee, liquid, ready-to-drink,soluble and dry coffee beverages, coffee beverage mixes and concentrates(syrups, pure, formulated, or in powder form; example of a “powder form”is a product comprising coffee, sweetener, and whitener all in powderform); tea, such as black, green, white, oolong, and flavored tea; andother hot drinks including flavor-, malt- or plant-based powders,granules, blocks or tablets mixed with milk or water.

The Snack Food category generally refers to any food that can be a lightinformal meal including, but not limited to Sweet and savory snacks andsnack bars. Examples of snack food include, but are not limited to fruitsnacks, chips/crisps, extruded snacks, tortilla/corn chips, popcorn,pretzels, nuts and other sweet and savory snacks. Examples of snack barsinclude, but are not limited to granola/muesli bars, breakfast bars,energy bars, fruit bars and other snack bars.

The Baked Goods category generally refers to any edible product theprocess of preparing which involves exposure to heat or excessivesunlight. Examples of baked goods include, but are not limited to bread,buns, cookies, muffins, cereal, toaster pastries, pastries, waffles,tortillas, biscuits, pies, bagels, tarts, quiches, cake, any bakedfoods, and any combination thereof.

The Ice Cream category generally refers to frozen dessert containingcream and sugar and flavoring. Examples of ice cream include, but arenot limited to: impulse ice cream; take-home ice cream; frozen yoghurtand artisanal ice cream; soy, oat, bean (e.g., red bean and mung bean),and rice-based ice creams.

The Confectionary category generally refers to edible product that issweet to the taste. Examples of confectionary include, but are notlimited to candies, gelatins, chocolate confectionery, sugarconfectionery, gum, and the likes and any combination products. The MealReplacement category generally refers to any food intended to replacethe normal meals, particularly for people having health or fitnessconcerns. Examples of meal replacement include, but are not limited toslimming products and convalescence products.

The Ready Meal category generally refers to any food that can be servedas meal without extensive preparation or processing. The read mealincludes products that have had recipe “skills” added to them by themanufacturer, resulting in a high degree of readiness, completion andconvenience. Examples of ready meal include, but are not limited tocanned/preserved, frozen, dried, chilled ready meals; dinner mixes;frozen pizza; chilled pizza; and prepared salads.

The Pasta and Noodle category includes any pastas and/or noodlesincluding, but not limited to canned, dried and chilled/fresh pasta; andplain, instant, chilled, frozen and snack noodles.

The Canned/Preserved Food category includes, but is not limited tocanned/preserved meat and meat products, fish/seafood, vegetables,tomatoes, beans, fruit, ready meals, soup, pasta, and othercanned/preserved foods.

The Frozen Processed Food category includes, but is not limited tofrozen processed red meat, processed poultry, processed fish/seafood,processed vegetables, meat substitutes, processed potatoes, bakeryproducts, desserts, ready meals, pizza, soup, noodles, and other frozenfood.

The Dried Processed Food category includes, but is not limited to rice,dessert mixes, dried ready meals, dehydrated soup, instant soup, driedpasta, plain noodles, and instant noodles.

The Chill Processed Food category includes, but is not limited tochilled processed meats, processed fish/seafood products, lunch kits,fresh cut fruits, ready meals, pizza, prepared salads, soup, fresh pastaand noodles.

The Sauces, Dressings and Condiments category includes, but is notlimited to tomato pastes and purees, bouillon/stock cubes, herbs andspices, monosodium glutamate (MSG), table sauces, soy based sauces,pasta sauces, wet/cooking sauces, dry sauces/powder mixes, ketchup,mayonnaise, mustard, salad dressings, vinaigrettes, dips, pickledproducts, and other sauces, dressings and condiments.

The Baby Food category includes, but is not limited to milk- orsoybean-based formula; and prepared, dried and other baby food.

The Spreads category includes, but is not limited to jams and preserves,honey, chocolate spreads, nut based spreads, and yeast based spreads.

The Dairy Product category generally refers to edible product producedfrom mammal's milk. Examples of dairy product include, but are notlimited to drinking milk products, cheese, yoghurt and sour milk drinks,and other dairy products.

Additional examples for comestible composition, particularly food andbeverage products or formulations, are provided as follows. Exemplarycomestible compositions include one or more confectioneries, chocolateconfectionery, tablets, countlines, bagged selflines/softlines, boxedassortments, standard boxed assortments, twist wrapped miniatures,seasonal chocolate, chocolate with toys, alfajores, other chocolateconfectionery, mints, standard mints, power mints, boiled sweets,pastilles, gums, jellies and chews, toffees, caramels and nougat,medicated confectionery, lollipops, liquorice, other sugarconfectionery, gum, chewing gum, sugarized gum, sugar free gum,functional gum, bubble gum, bread, packaged/industrial bread,unpackaged/artisanal bread, pastries, cakes, packaged/industrial cakes,unpackaged/artisanal cakes, cookies, chocolate coated biscuits, sandwichbiscuits, filled biscuits, savory biscuits and crackers, breadsubstitutes, breakfast cereals, rte cereals, family breakfast cereals,flakes, muesi, other cereals, children's breakfast cereals, hot cereals,ice cream, impulse ice cream, single portion dairy ice cream, singleportion water ice cream, multi pack dairy ice cream, multi pack waterice cream, take home ice cream, take home dairy ice cream, ice creamdesserts, bulk ice cream, take home water ice cream, frozen yoghurt,artisanal ice cream, dairy products, milk, fresh/pasteurized milk, fullfat fresh/pasteurized milk, semi skimmed fresh/pasteurized milk, longlife/uht milk, full fat long life/uht milk, semi skimmed long life/uhtmilk, fat free long life/uht milk, goat milk, condensed/evaporated milk,plain condensed/evaporated milk, flavored, functional and othercondensed milk, flavored milk drinks, dairy only flavored milk drinks,flavored milk drinks with fruit juice, soy milk, sour milk drinks,fermented dairy drinks, coffee whiteners (e.g., dairy and non-dairybased creamers or whiteners for coffee beverages), powder milk, flavoredpowder milk drinks, cream, cheese, processed cheese, spreadableprocessed cheese, unspreadable processed cheese, unprocessed cheese,spreadable unprocessed cheese, hard cheese, packaged hard cheese,unpackaged hard cheese, yoghurt, plain/natural yoghurt, flavoredyoghurt, fruited yoghurt, probiotic yoghurt, drinking yoghurt, regulardrinking yoghurt, probiotic drinking yoghurt, chilled and shelf stabledesserts, dairy based desserts, soy based desserts, chilled snacks,fromage frais and quark, plain fromage frais and quark, flavored fromagefrais and quark, savory fromage frais and quark, sweet and savorysnacks, fruit snacks, chips/crisps, extruded snacks, tortilla/cornchips, popcorn, pretzels, nuts, other sweet and savory snacks, snackbars, granola bars, breakfast bars; energy bars, fruit bars, other snackbars, meal replacement products, slimming products, convalescencedrinks, ready meals, canned ready meals, frozen ready meals, dried readymeals, chilled ready meals, dinner mixes, frozen pizza, chilled pizza,soup, canned soup, dehydrated soup, instant soup, chilled soup, hotsoup, frozen soup, pasta, canned pasta, dried pasta, chilled/freshpasta, noodles, plain noodles, instant noodles, cups/bowl instantnoodles, pouch instant noodles, chilled noodles, snack noodles, cannedfood, canned meat and meat products, canned fish/seafood, cannedvegetables, canned tomatoes, canned beans, canned fruit, canned readymeals, canned soup, canned pasta, other canned foods, frozen food,frozen processed red meat, frozen processed poultry, frozen processedfish/seafood, frozen processed vegetables, frozen meat substitutes,frozen potatoes, oven baked potato chips, other oven baked potatoproducts, non-oven frozen potatoes, frozen bakery products, frozendesserts, frozen ready meals, frozen pizza, frozen soup, frozen noodles,other frozen food, dried food, dessert mixes, dried ready meals,dehydrated soup, instant soup, dried pasta, plain noodles, instantnoodles, cups/bowl instant noodles, pouch instant noodles, chilled food,chilled processed meats, chilled fish/seafood products, chilledprocessed fish, chilled coated fish, chilled smoked fish, chilled lunchkit, chilled ready meals, chilled pizza, chilled soup, chilled/freshpasta, chilled noodles, oils and fats, olive oil, vegetable and seedoil, cooking fats, butter, margarine, spreadable oils and fats,functional spreadable oils and fats, sauces, dressings and condiments,tomato pastes and purees, bouillon/stock cubes, stock cubes, gravygranules, liquid stocks and fonds, herbs and spices, fermented sauces,soy based sauces, pasta sauces, wet sauces, dry sauces/powder mixes,ketchup, mayonnaise, regular mayonnaise, mustard, salad dressings,regular salad dressings, low fat salad dressings, vinaigrettes, dips,pickled products, other sauces, dressings and condiments, baby food,milk formula, standard milk formula, follow on milk formula, toddlermilk formula, hypoallergenic milk formula, prepared baby food, driedbaby food, other baby food, spreads, jams and preserves, honey,chocolate spreads, nut based spreads, and yeast-based spreads. Examplesof comestible compositions also include confectioneries, bakeryproducts, ice creams, dairy products, sweet and savory snacks, snackbars, meal replacement products, ready meals, soups, pastas, noodles,canned foods, frozen foods, dried foods, chilled foods, oils and fats,baby foods, or spreads or a mixture thereof. Examples of comestiblecompositions also include breakfast cereals, sweet beverages or solid orliquid concentrate compositions for preparing beverages. Examples ofcomestible compositions also include coffee flavored food (e.g., coffeeflavored ice cream).

Values expressed in a range format should be interpreted in a flexiblemanner to include not only the numerical values explicitly recited asthe limits of the range, but also to include all the individualnumerical values or sub-ranges encompassed within that range as if eachnumerical value and sub-range were explicitly recited. For example, arange of “about 0.1% to about 5%” or “about 0.1% to 5%” should beinterpreted to include not just about 0.1% to about 5%, but also theindividual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g.,0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range.The statement “about X to Y” has the same meaning as “about X to aboutY,” unless indicated otherwise. Likewise, the statement “about X, Y, orabout Z” has the same meaning as “about X, about Y, or about Z,” unlessindicated otherwise.

In this document, the terms “a,” “an,” or “the” are used to include oneor more than one unless the context clearly dictates otherwise. The term“or” is used to refer to a nonexclusive “or” unless otherwise indicated.In addition, it is to be understood that the phraseology or terminologyemployed herein, and not otherwise defined, is for the purpose ofdescription only and not of imitation. Any use of section headings isintended to aid reading of the document and is not to be interpreted aslimiting; information that is relevant to a section heading may occurwithin or outside of that particular section. Furthermore, allpublications, patents, and patent documents referred to in this documentare incorporated by reference herein in their entirety, as thoughindividually incorporated by reference. In the event of inconsistentusages between this document and those documents so incorporated byreference, the usage in the incorporated reference should be consideredsupplementary to that of this document; for irreconcilableinconsistencies, the usage in this document controls.

In the methods described herein, the steps can be carried out in anyorder without departing from the principles of the invention, exceptwhen a temporal or operational sequence is explicitly recited.Furthermore, specified steps can be carried out concurrently unlessexplicit claim language recites that they be carried out separately. Forexample, a claimed step of doing X and a claimed step of doing Y can beconducted simultaneously within a single operation, and the resultingprocess will fall within the literal scope of the claimed process.

The term “about” as used herein can allow for a degree of variability ina value or range, for example, within 10%, within 5%, or within 1% of astated value or of a stated limit of a range.

The term “substantially” as used herein refers to a majority of, ormostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more.

EXAMPLES

The present invention can be better understood by reference to thefollowing examples which are offered by way of illustration. The presentinvention is not limited to the examples given herein.

Example 1

Samples were prepared via the design shown in Table 1, in weight tovolume percentage. An appropriate amount of steviol glycoside (SG) wasweighed into a 10 mL glass vial and diluted with an appropriate volumeof pH 4 citrate buffer, e.g., for 0.6% level, 27 mg was diluted into 4.5mL of buffer. This was repeated for all conditions in Table 1. Samplesthat were designed for pH 2.5 were then adjusted via phosphoric acid andpH meter to pH 2.5, dropwise. For these samples, the same lot of steviolglycoside stabilizing compound (SC) was used, which was purified fromstevia leaves. Two different SG sources were used, RM80 (>80% Reb M on adry weight basis) and RA95 (>95% Reb A on a dry weight basis).

At each time point, the solutions were centrifuged at 10,000 rpm for twominutes to remove any insoluble material from the analysis (even thoughnone was visible). An aliquot of the supernatant was diluted into 55%methanol for analysis by UHPLC-UV. The chromatographic analysis wasperformed on a C18-based reversed-phase chromatography column atelevated temperature under gradient conditions, utilizingtrifluoroacetic acid in water and acetonitrile. SGs were detectedutilizing a UV detector set to 210 nm. A linear calibration curve wasapplied using a high-purity (>99%) Reb A standard as a referencesolution.

TABLE 1 SG % SG Type SC % Storage Cond pH Times (weeks) # of Pulls # ofReplicates 0.6% RM80 0.6% RT 2.5 0, 14, 26, 52 4 3 1.5% RM80 1.5% RT 2.50, 4, 14, 26, 39, 52 6 1 3.0% RM80 3.0% RT 2.5 0, 4, 14, 26, 39, 52 6 33.0% RM80 4.5% RT 2.5 0, 4, 14, 26, 39, 52 6 3 6.0% RM80 6.0% RT 2.5 0,4, 14, 26, 39, 52 6 1 6.0% RA95 6.0% RT 2.5 0, 4, 14, 26, 39, 52 6 30.6% RM80 0.6% 4C 2.5 14, 26, 52 3 1 1.5% RM80 1.5% 4C 2.5 4, 14, 26,39, 52 5 3 3.0% RM80 3.0% 4C 2.5 4, 14, 26, 39, 52 5 1 3.0% RM80 4.5% 4C2.5 4, 14, 26, 39, 52 5 1 6.0% RM80 6.0% 4C 2.5 4, 14, 26, 39, 52 5 36.0% RA95 6.0% 4C 2.5 4, 14, 26, 39, 52 5 1 0.6% RM80 0.6% RT 4.0 0, 14,26, 52 4 3 1.5% RM80 1.5% RT 4.0 0, 4, 14, 26, 39, 52 6 1 3.0% RM80 3.0%RT 4.0 0, 4, 14, 26, 39, 52 6 3 3.0% RM80 4.5% RT 4.0 0, 4, 14, 26, 39,52 6 3 6.0% RM80 6.0% RT 4.0 0, 4, 14, 26, 39, 52 6 1 35.0% RM80 35.0%RT 4.0 0, 26, 40, 52 4 1 6.0% RA95 6.0% RT 4.0 0, 4, 14, 26, 39, 52 6 30.6% RM80 0.6% 4C 4.0 14, 26, 52 3 1 1.5% RM80 1.5% 4C 4.0 4, 14, 26,39, 52 5 3 3.0% RM80 3.0% 4C 4.0 4, 14, 26, 39, 52 5 1 3.0% RM80 4.5% 4C4.0 4, 14, 26, 39, 52 5 1 6.0% RM80 6.0% 4C 4.0 4, 14, 26, 39, 52 5 36.0% RA95 6.0% 4C 4.0 4, 14, 26, 39, 52 5 1

Briefly, long term storage chemical stability data stored at 4° C.; roomtemperature (˜22° C.); at pH 4; and at pH 2.5>94% recovery of the SGafter 48+ weeks of storage. The long term storage chemical stabilitydata is given in Table 2. A value of NM denotes that no measurement wastaken at that time.

TABLE 2 Time Experiment (weeks) 0 4 14 26 39 40 48 6% RA95 Reb A % 100.099.5 98.3 98.3 98.6 NM 98.5 with 6% Recovery SCs at 4 C. and pH 2.5 6%RA95 Reb A % 100.0 98.8 99.1 98.0 98.7 NM 98.6 with 6% Recovery SCs at 4C. and pH 4 6% RA95 Reb A % 100.0 99.5 98.6 98.2 98.0 NM 97.8 with 6%Recovery SCs at RT and pH 2.5 6% RA95 Reb A % 100.0 99.4 98.4 98.4 98.3NM 98.3 with 6% Recovery SCs at RT and pH 4 0.6% Reb M % 100.0 NM 102.4102.4 NM NM 98.0 RM80 with Recovery 0.6% SCs at 4 C. and pH 2.5 0.6% RebM % 100.0 NM 102.8 102.6 NM NM 99.0 RM80 with Recovery 0.6% SCs at 4 C.and pH 4 0.6% Reb M % 100.0 NM 101.1 100.2 NM NM 96.0 RM80 with Recovery0.6% SCs at RT and pH 2.5 0.6% Reb M % 100.0 NM 101.7 101.5 NM NM 99.2RM80 with Recovery 0.6% SCs at RT and pH 4 1.5% Reb M % 100.0 102.6102.6 102.5 98.5 NM 97.9 RM80 with Recovery 1.5% SCs at 4 C. and pH 2.51.5% Reb M % 100.0 102.7 102.5 102.9 98.8 NM 98.7 RM80 with Recovery1.5% SCs at 4 C. and pH 4 1.5% Reb M % 100.0 101.8 100.8 98.6 97.3 NM95.3 RM80 with Recovery 1.5% SCs at RT and pH 2.5 1.5% Reb M % 100.0102.1 101.5 101.8 99.2 NM 98.3 RM80 with Recovery 1.5% SCs at RT and pH4 3% RM80 Reb M % 100.0 102.5 102.2 102.1 98.5 NM 97.9 with 3% RecoverySCs at 4 C. and pH 2.5 3% RM80 Reb M % 100.0 102.6 102.1 102.5 98.9 NM98.5 with 3% Recovery SCs at 4 C. and pH 4 3% RM80 Reb M % 100.0 101.8101.0 99.8 95.9 NM 95.0 with 3% Recovery SCs at RT and pH 2.5 3% RM80Reb M % 100.0 102.0 101.6 101.2 98.2 NM 97.7 with 3% Recovery SCs at RTand pH 4 3% RM80 Reb M % 100.0 103.5 103.4 103.3 98.3 NM 96.7 with 4.5%Recovery SCs at 4 C. and pH 2.5 3% RM80 Reb M % 100.0 103.4 102.8 103.697.4 NM 97.7 with 4.5% Recovery SCs at 4 C. and pH 4 3% RM80 Reb M %100.0 102.7 101.5 100.6 96.2 NM 94.0 with 4.5% Recovery SCs at RT and pH2.5 3% RM80 Reb M % 100.0 103.4 102.3 102.2 98.0 NM 97.7 with 4.5%Recovery SCs at RT and pH 4 6% RM80 Reb M % 100.0 102.0 102.0 101.8 97.9NM 97.4 with 6% Recovery SCs at 4 C. and pH 2.5 6% RM80 Reb M % 100.0102.0 101.6 102.2 98.3 NM 97.7 with 6% Recovery SCs at 4 C. and pH 4 6%RM80 Reb M % 100.0 101.6 100.7 99.5 95.0 NM 94.1 with 6% Recovery SCs atRT and pH 2.5 6% RM80 Reb M % 100.0 101.6 101.2 100.8 97.2 NM 96.7 with6% Recovery SCs at RT and pH 4 35% RM80 Reb M % 100.0 NM NM 99.5 NM 95.996.2 with 35% Recovery SCs at RT and pH 4

This example shows that the steviol glycoside stability compounds areeffective to stabilize steviol glycoside over time. Steviol glycosidestability compounds are effective to stabilize steviol glycoside over 48weeks with greater than 94% recovery of the steviol glycoside at 4° C.,at room temperature, at pH 4, and/or at pH 2.5.

Example 2

Samples were prepared at 500 ppm (a use level that can be used inbeverages) for storage at room temperature (RT, which is −22° C.). A pH1.7 buffer (Oakton, part number 00654-01) was purchased from Fisher andused to dilute all samples. The lot of SC material used in the study wasderived from yerba mate. Three total solutions were made for the study:one with no additives (500 ppm Reb M in pH 1.7 buffer), one with 500 ppmReb M and 500 ppm SCs, and one negative control with 500 ppm Reb M and500 ppm ascorbic acid (a common antioxidant).

Highly purified Reb M (>99%) was weighed directly into a 40 mL glassvial at an appropriate level, so the final concentration was 500 ppm,i.e., 20 mg into 40 mL. Next the additive (if present) was weigheddirectly in the same vial. Finally the total volume of pH 1.7 buffer wasadded and the solution was mixed to dissolve.

At each time point, the solutions were centrifuged at 10,000 rpm for twominutes to remove any insoluble material from the analysis, even thoughnone was visible. An aliquot of the supernatant was injected directlyfor analysis by UHPLC-UV. The chromatographic analysis was performed ona C18-based reversed-phase chromatography column at elevated temperatureunder gradient conditions, utilizing trifluoroacetic acid in water andacetonitrile. SGs were detected utilizing a UV detector set to 210 nm. Alinear calibration curve was applied using a high-purity (>99%) Reb Astandard as a reference solution.

The results presented in Table 3 demonstrate that SCs generally improvethe chemical stability of SGs, even at relatively low levels, such as atthe use level.

TABLE 3 Experiment Time (Days) 0 7 14 24 35 Reb M pH 1.7 % Reb M 100.093.4 87.7 80.2 73.0 Recovery Reb M pH 1.7 % Reb M 100.0 93.6 88.0 80.573.5 with AA Recovery Reb M pH 1.7 % Reb M 100.0 94.0 88.7 81.4 74.6with SCs Recovery

Example 3

Twelve total solutions were made for this study: 0.1% RA95, RM80, or RebM with no additives; 0.1% RA95, RM80, or Reb M with 0.1% SCs; 1% RA95,RM80, or Reb M with 1% SCs; and 5% RA95, RM80, or Reb M with 5% SCs. Toprepare each solution RA95, RM80, or highly purified Reb M (>99%) wasweighed into a glass vial in the appropriate amount followed by SCmaterial (derived from yerba mate). Samples were diluted in water andheated at 80° C. to solubilize the glycosides. After allowing solutionsto cool to room temperature, phosphoric acid was added to give a finalconcentration of 5% phosphoric acid. Samples were stored at 40° C. forthe duration of the study.

At each time point, the solutions were centrifuged at 10,000 rpm for twominutes to remove any insoluble material from the analysis (even thoughnone was visible). An aliquot of the supernatant was diluted into waterfor analysis by UHPLC-UV. The chromatographic analysis was performed ona C18-based reversed-phase chromatography column at elevated temperatureunder gradient conditions, utilizing trifluoroacetic acid in water andacetonitrile. SGs were detected utilizing a UV detector set to 210 nm. Alinear calibration curve was applied using a high-purity (>99%) Reb Astandard as a reference solution. Table 4 shows the percent recoverydata from RA95 (>99%) study in 5% phosphoric acid matrix (pH<<1) storedat 40° C. And Table 5 shows the percent recovery data from pure Reb M(>99%) study in 5% phosphoric acid matrix (pH<<1) stored at 40° C.Finally, Table 6 shows the percent recovery data from Reb M in RM80product in 5% phosphoric acid matrix (pH<<1) stored at 40° C.

TABLE 4 Experiment Time (Days) 0 1 2 3 7 0.1% RA95 No % Reb A 100.0 57.734.4 22.2 6.58 SCs Recovery 0.1% RA95 0.1% % Reb A 100.0 58.3 35.6 23.16.68 SCs Recovery 1% RA95 1% % Reb A 100.0 73.8 55.5 42.6 16.2 SCsRecovery 5% RA95 5% % Reb A 100.0 89.7 81.1 73.4 50.0 SCs Recovery

TABLE 5 Experiment Time (Days) 0 1 2 3 7 0.1% Reb M No % Reb M 100.055.1 31.5 19.9 5.07 SCs Recovery 0.1% Reb M % Reb M 100.0 57.9 34.2 21.96.28 0.1% SCs Recovery 1% Reb M 1% % Reb M 100.0 71.8 51.9 38.5 13.4 SCsRecovery 5% Reb M 5% % Reb M 100.0 85.3 73.0 62.9 36.1 SCs Recovery

TABLE 6 Experiment Time (Days) 0 1 2 3 7 0.1% RM80 with 0% % Reb M 100.060.1 35.8 22.1 4.36 SCs and 5% H3PO4 Recovery 0.1% RM80 with % Reb M100.0 62.2 37.9 24.1 5.07 0.1% SCs and 5% Recovery H3PO4 1% RM80 with 1%% Reb M 100.0 77.0 58.3 45.4 17.0 SCs and 5% H3PO4 Recovery 5% RM80 with5% % Reb M 100.0 91.2 83.0 75.2 49.4 SCs and 5% H3PO4 Recovery

The results in Tables 4-6 demonstrate that the steviol glycosidestability is concentration-dependent. That is, the higher theconcentration of steviol glycoside (SG) and steviol glycosidestabilizing compound, the more stable the steviol glycoside. Thus, forexample, at 5 wt. % steviol glycoside and 5 wt. % steviol glycosidestabilizing compound, one observes a significant increase in thestability of the SG as compared to a lower concentration solution.

Example 4

Three solutions were made for this study: 0.1% RM80 with no additives,0.1% RM80 with 0.1% SCs, and 0.1% RM80 with 0.3% SCs. To prepare eachsolution, RM80 was weighed into a glass vial in the appropriate amountfollowed by SC material (derived from yerba mate). Samples were dilutedin water and vortexed to solubilize the glycosides. Phosphoric acid wasadded to give a final concentration of 0.1% phosphoric acid. Sampleswere stored at room temperature (20-24° C.) for the duration of thestudy.

At each time point, the solutions were centrifuged at 10,000 rpm for twominutes to remove any insoluble material from the analysis (even thoughnone was visible). An aliquot of the supernatant was diluted into waterfor analysis by UHPLC-UV. The chromatographic analysis was performed ona C18-based reversed-phase chromatography column at elevated temperatureunder gradient conditions, utilizing trifluoroacetic acid in water andacetonitrile. SGs were detected utilizing a UV detector set to 210 nm. Alinear calibration curve was applied using a high-purity (>99%) Reb Astandard as a reference solution. Table 7 shows data from a use level(e.g., 0.1% RM80) study in 0.1% phosphoric acid matrix at roomtemperature (˜22° C.). Table 8 is the statistical evaluation of thestability enhancement at each timepoint compared to the solution withoutstability enhancing compounds.

TABLE 7 Time Experiment (Days) 0 9 16 30 37 51 58 65 0.1% RM80 witoutSCs % Reb M 100.0 97.8 96.6 93.6 91.5 89.6 88.1 86.2 and 0.1% H3PO4Recovery 0.1% RM80 with 0.1% % Reb M 100.0 99.6 98.0 94.5 93.5 91.4 90.990.1 SCs and 0.1% H3PO4 Recovery 0.1% RM80 with 0.3% % Reb M 100.0 98.997.5 94.0 93.3 91.0 91.1 89.9 SCs and 0.1% H3PO4 Recovery

TABLE 8 Time Experiment (Days) 0 9 16 30 37 51 58 65 0.1% RM80 witoutSCs % Reb M — — — — — — — — and 0.1% H3PO4 Recovery 0.1% RM80 with 0.1%% Reb M — 0.0002 0.004 0.02 0.01 0.002 0.004 0.0003 SCs and 0.1% H3PO4Recovery 0.1% RM80 with 0.3% % Reb M — 0.01 0.02 0.3 0.001 0.06 0.0020.0009 SCs and 0.1% H3PO4 Recovery p-values comparing % reb M remainingas compared to the respective timepoint without SCs present.

These results demonstrate that even at use levels, SCs provide SGsprotection from acid hydrolysis. The results also demonstrate thatadditional SCs beyond the concentration needed to complex all of the SGscan have no additional benefit. For example, 0.3% SCs has the sameprotective effects as 0.1% SCs.

Example 5

Four solutions were made for this study using Rosmarinic Acid: 0.1% RebM with no additives; 0.1% Reb M with 0.1% SCs; 1% Reb M with 1% SCs; and5% Reb M with 5% SCs. Four solutions were made for this study usingCichoric Acid: 0.1% Reb M with no additives; 0.1% Reb M with 0.1% SCs;1% Reb M with 1% SCs; and 5% Reb M with 5% SCs. To prepare each solutionhighly purified Reb M (>99%) was weighed into a glass vial in theappropriate amount followed by SC material (either Rosmarinic Acid orCichoric Acid). Samples were diluted in water and heated at 80° C. tosolubilize the glycosides. After allowing solutions to cool to roomtemperature, phosphoric acid was added to give a final concentration of5% phosphoric acid. Samples were stored at 40° C. for the duration ofthe study.

At each time point, the solutions were centrifuged at 10,000 rpm for twominutes to remove any insoluble material from the analysis (even thoughnone was visible). An aliquot of the supernatant was diluted into waterfor analysis by UHPLC-UV. The chromatographic analysis was performed ona C18-based reversed-phase chromatography column at elevated temperatureunder gradient conditions, utilizing trifluoroacetic acid in water andacetonitrile. SGs were detected utilizing a UV detector set to 210 nm. Alinear calibration curve was applied using a high-purity (>99%) Reb Mstandard as a reference solution.

Tables 9 and 10 show the percent recovery data from Reb M (>99%) studyin 5% phosphoric acid matrix (pH<<1) stored at 40° C., in the presenceof Rosmarinic Acid and Cichoric Acid respectively.

TABLE 9 Experiment Time (Days) 0 1 2 3 7 0.1% Reb M No % Reb M 98 56 3320 6 SCs Recovery 0.1% Reb M % Reb M 97 59 57 24 7 0.1% SCs Recovery 1%Reb M 1% % Reb M 98 73 54 41 16 SCs Recovery 5% Reb M 5% % Reb M 97 8166 55 27 SCs Recovery

TABLE 10 Experiment Time (Days) 0 1 2 3 7 0.1% Reb M No % Reb M 98 56 3320 6 SCs Recovery 0.1% Reb M % Reb M 97 59 36 23 6 0.1% SCs Recovery 1%Reb M 1% % Reb M 98 72 54 40 13 SCs Recovery 5% Reb M 5% % Reb M 97 7963 13 23 SCs Recovery

Example 6

The steviol glycoside stabilzing compounds are themselves subject todegradation over time. The SCs can hydrolyze under acidic conditions toform caffeic acid. The SCs can also oxidize over time when exposed tooxygen. To study acidic degradation, the SCs were also quantified in thesame experiment as outlined in Table 11. The SCs are much more resistantto acidic hydrolysis than the SGs, but the stability enhancement followsthe same trend. At higher concentrations, the SGs and SCs both are moreefficiently stabilized.

TABLE 11 Experiment Time (Days) 0 1 2 3 7 0.1% Reb M No % SC — — — — —SCs Recovery 0.1% Reb M % SC 100% 99% 98% 98% 95% 0.1% SCs Recovery 1%Reb M 1% % SC 100% 99% 99% 99% 97% SCs Recovery 5% Reb M 5% % SC 100%100%  99% 99% 99% SCs Recovery

Example 7

To study the oxidative stability of the SEs, a solution of 500 ppm SCwas prepared in pH 7 buffer. A second solution of 500 ppm SC with 500ppm RM80 was prepared in pH 7 buffer. Both of these solutions werestirred aggressively and exposed to the oxygen in the atmosphere for 72days. The results are summarized in Table 12 below and demonstrate thatthe presence of SGs will slow the oxidative degradation of the SCs.

TABLE 12 Experiment Time (Days) 0 72 0.05% SCs % SC Recovery 100% 17%0.05% SCs % SC Recovery 100% 48% 0.05% RM80

Example 8

It has been hypothesized that steviol glycosides (SGs) and steviolglycoside stabilizing compounds (SCs) will form a tight-binding complexin solution. If this is true, the magnetic environment of the complexwould be substantially different than of the individual compoundsdissolved in water. This would result in substantial shifting (Δδ>0.02ppm) in their respective ¹H NMR spectra.

A total of four samples were prepared for this study, and they arelisted below. Each sample was dissolved fully in water, with or withoutheat as noted below, flash frozen at −80° C., and then placed on alyophilizer until dry. The dry powders were subsequently dissolved inD₂O at room temperature and analyzed by ‘H and’3C NMR. The concentrationof Sample 1 is substantially lower than Samples 2-4, as Reb M solubilityin D₂O is much lower when SCs are not present, and this resulted inrelatively poor quality spectra.

Sample 1: 10 mg Reb M in 1 mL water—heated in H₂O

Sample 2: 10 mg SE in 1 mL water—heated in H₂O

Sample 3: 10 mg Reb M+10 mg SE in 1 mL water—heated in H₂O

Sample 4: 10 mg Reb M+10 mg SE in 3 mL water—not heated in H₂O

Using the following numbering convention for SC molecules:

in this case, monocaffeoylquinic acid and dicaffeoylquinic acid. Theobserved signals are the sum of the mixture of isomers. The ¹H NMR dataare shown in Tables 13 (showing ¹H NMR data with significant shifting inthe caffeic acid moieties of the SCs) and 14 (Showing ¹H NMR data withsignificant shifting in the steviol core of the SG).

TABLE 13 Average Average Shifting Shifting Protons δ ppm range Sample 3vs. 2 Sample 4 vs. 2 C7′ and C7″ 7.5-7.7 +0.034 and +0.029 and +0.053+0.045 C2′ and C2″ 7.15-7.2  +0.039 +0.034 C6′ and C6″ 7.05-7.15 +0.030+0.024 C5′ and C5″ 6.9-7.0 +0.022 +0.018 C8′ and C8″ 6.3-6.5 +0.033 and+0.028 and +0.055 +0.047

TABLE 14 Average Average Shifting Shifting Protons δ ppm range Sample 3vs 1 Sample 4 vs 1 C20 Methyl 0.90 −0.13 −0.15 C18 Methyl 1.27 −0.09−0.10

There is a substantial amount of shifting in both the SC signals and theSG signals when the mixture of molecules is present. This shifting issimilar when the compounds are heated in water together versus when theyare mixed at room temperature, but slightly greater when heated. Themoieties which show the strongest shifting are the caffeic acid moietiesof the SCs and the steviol backbone of the SGs, suggesting a stronginteraction between the most hydrophobic regions of each molecule,leaving the glucose and qunic acid moieties free to interact with water,thus possibly increasing the stability of SGs and SCs.

The present invention provides for the following embodiments, thenumbering of which is not to be construed as designating levels ofimportance:

Embodiment 1 relates to a composition comprising:

-   -   a steviol glycoside; and    -   a steviol glycoside stabilizing compound in an amount effective        to    -   reduce degradation of the steviol glycoside;    -   wherein the steviol glycoside stabilizing compound is at least        one    -   compound, and isomers thereof, selected from the group        consisting of:        -   caffeic acid, an ester of caffeic acid, an ester of caffeic            acid and quinic acid, an ester of caffeic acid and quinic            acid comprising a single caffeic acid moiety (e.g.,            chlorogenic, cryptochlorogenic, and neochlorogenic acid;            structures of each are provided herein), an ester of caffeic            acid and quinic acid comprising more than one caffeic acid            moiety (e.g., 1,3-dicaffeoylquinic acid,            1,4-dicaffeoylquinic acid, 1,5-dicaffeoylquinic acid,            3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and            4,5-dicaffeoylquinic acid; structures of each are provided            herein);        -   feruic acid, an ester of ferulic acid, an ester of ferulic            acid and quinic acid, an ester of ferulic acid and quinic            acid comprising a single feruic acid moiety, an ester of            ferulic acid and quinic acid comprising more than one feruic            acid moiety;        -   3-(3,4-dihydroxyphenyl)lactic acid, a            3-(3,4-dihydroxyphenyl)lactic acid derivative, an ester of            3-(3,4-dihydroxyphenyl)lactic acid, an ester of a            3-(3,4-dihydroxyphenyl)lactic acid derivative,        -   quinic acid, a quinic acid derivative, an ester of quinic            acid, an ester of a quinic acid derivative;        -   p-coumaric acid, an ester of p-coumaric acid, an ester of            p-coumaric acid and quinic acid, an ester of p-coumaric acid            and quinic acid comprising a single p-coumaric acid moiety,            an ester of p-coumaric acid and quinic acid comprising more            than one p-coumaric acid moiety;        -   sinapic acid, an ester of sinapic acid, an ester of sinapic            acid and quinic acid, an ester of sinapic acid and quinic            acid comprising a single sinapic acid moiety, an ester of            sinapic acid and quinic acid comprising more than one            sinapic acid moiety;        -   tartaric acid, a tartaric acid derivative, an ester of            tartaric acid, an ester of a tartaric acid derivative, and            3-O-feruloylquinic acid, 4-O-feruloylquinic acid,            5-O-feruloylquinic acid, 3,4-diferuloylquinic acid,            3,5-diferuloylquinic acid, 4,5-diferuloylquinic acid.

Embodiment 2 relates to the composition of Embodiment 1, wherein thecomposition is an aqueous composition.

Embodiment 3 relates to the composition of Embodiments 1-2, wherein theamount of steviol glycoside stabilizing compound effective to reducedegradation of the steviol glycoside is an amount such that at leastabout 10 wt. % of an initial steviol glycoside remains when thestabilized steviol glycoside composition is subjected to storage for 7days at 40° C. in 5% phosphoric acid.

Embodiment 4 relates to the composition of Embodiments 1-3, wherein thesteviol glycoside comprises at least about 0.03 wt. % steviol glycoside.

Embodiment 5 relates to the composition of Embodiments 1-3, wherein thesteviol glycoside comprises at least about 0.6 wt % steviol glycoside.

Embodiment 6 relates to the composition of Embodiments 1-5, wherein thecomposition comprises a 1:0.3 to 1:3 ratio by weight of steviolglycoside to steviol glycoside stabilizing compound.

Embodiment 7 relates to the composition of Embodiments 1-6, wherein thecomposition has a pH of less than about 4.

Embodiment 8 relates to the composition of Embodiments 1-6, wherein thecomposition has a pH of less than about 1.

Embodiment 9 relates to the composition of Embodiments 1-8, wherein thecomposition is stored at room temperature.

Embodiment 10 relates to the composition of Embodiments 1-8, wherein thecomposition is stored at about 4° C.

Embodiment 11 relates to the composition of Embodiments 1-10, whereinthe steviol glycoside is Rebaudioside A or Rebaudioside M.

Embodiment 12 relates to a beverage concentrate product comprising thecomposition of Embodiments 1-11, wherein the steviol glycoside comprisesbetween about 1,800 ppm and about 10,000 ppm steviol glycoside.

Embodiment 13 relates to a liquid water enhancer product comprising thecomposition of Embodiments 1-11, wherein the steviol glycoside comprisesbetween about 1.5 wt. % and about 3.5 wt. %. steviol glycoside.

Embodiment 14 relates to a liquid sweetener comprising the compositionof Embodiments 1-11, wherein, the steviol glycoside comprises betweenabout 1.0 wt. % and about 10 wt. % steviol glycoside.

1. A composition comprising: a steviol glycoside; and a steviolglycoside stabilizing compound in an amount effective to reducedegradation of the steviol glycoside; wherein the steviol glycosidestabilizing compound is at least one compound selected from the groupconsisting of the following and isomers thereof: caffeic acid, an esterof caffeic acid, an ester of caffeic acid and quinic acid, an ester ofcaffeic acid and quinic acid comprising a single caffeic acid moiety(e.g., chlorogenic, cryptochlorogenic, and neochlorogenic acid;structures of each are provided herein), an ester of caffeic acid andquinic acid comprising more than one caffeic acid moiety (e.g.,1,3-dicaffeoylquinic acid, 1,4-dicaffeoylquinic acid,1,5-dicaffeoylquinic acid, 3,4-dicaffeoylquinic acid,3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid; structures ofeach are provided herein); ferulic acid, an ester of ferulic acid, anester of ferulic acid and quinic acid, an ester of ferulic acid andquinic acid comprising a single ferulic acid moiety, an ester of ferulicacid and quinic acid comprising more than one ferulic acid moiety;3-(3,4-dihydroxyphenyl)lactic acid, a 3-(3,4-dihydroxyphenyl)lactic acidderivative, an ester of 3-(3,4-dihydroxyphenyl)lactic acid, an ester ofa 3-(3,4-dihydroxyphenyl)lactic acid derivative, quinic acid, a quinicacid derivative, an ester of quinic acid, an ester of a quinic acidderivative; p-coumaric acid, an ester of p-coumaric acid, an ester ofp-coumaric acid and quinic acid, an ester of p-coumaric acid and quinicacid comprising a single p-coumaric acid moiety, an ester of p-coumaricacid and quinic acid comprising more than one p-coumaric acid moiety;sinapic acid, an ester of sinapic acid, an ester of sinapic acid andquinic acid, an ester of sinapic acid and quinic acid comprising asingle sinapic acid moiety, an ester of sinapic acid and quinic acidcomprising more than one sinapic acid moiety; tartaric acid, a tartaricacid derivative, an ester of tartaric acid, an ester of a tartaric acidderivative, and 3-O-feruloylquinic acid, 4-O-feruloylquinic acid,5-O-feruloylquinic acid, 3,4-diferuloylquinic acid, 3,5-diferuloylquinicacid, 4,5-diferuloylquinic acid.
 2. The composition of claim 1, whereinthe composition is an aqueous composition.
 3. The composition of claim1, wherein the amount of steviol glycoside stabilizing compoundeffective to reduce degradation of the steviol glycoside is an amountsuch that at least about 10 wt. % of an initial steviol glycosideremains when the stabilized steviol glycoside composition is subjectedto storage for 7 days at 40° C. in 5% phosphoric acid.
 4. Thecomposition of claim 1, wherein the steviol glycoside comprises at leastabout 0.03 wt. % of the composition.
 5. The composition of claim 1,wherein the steviol glycoside comprises at least about 0.6 wt. % of thecomposition.
 6. The composition of claim 1, wherein the compositioncomprises a 1:0.3 to 1:3 ratio by weight of steviol glycoside to steviolglycoside stabilizing compound.
 7. The composition of claim 1, whereinthe composition has a pH of less than about
 4. 8. The composition ofclaim 1, wherein the composition has a pH of less than about
 1. 9. Thecomposition of claim 1, wherein the composition is stored at roomtemperature.
 10. The composition of claim 1, wherein the composition isstored at about 4° C.
 11. The composition of claim 1, wherein thesteviol glycoside is Rebaudioside A or Rebaudioside M.
 12. A beverageconcentrate product comprising the composition of claim 1, wherein thebeverage concentrate product comprises between about 1,800 ppm and about10,000 ppm of the steviol glycoside.
 13. A liquid water enhancer productcomprising the composition of claim 1, wherein the liquid water enhancerproduct comprises between about 1.5 wt. % and about 3.5 wt. % of thesteviol glycoside.
 14. A liquid sweetener comprising the composition ofclaim 1, wherein, the liquid sweetener comprises between about 1.0 wt. %and about 10 wt. % of the steviol glycoside.